Crystalline form of 3-((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)propanenitrile

ABSTRACT

The invention provides a crystalline form of the compound of formula: 
                         
that is an inhibitor of JAK kinases. The invention also provides pharmaceutical compositions comprising such crystalline form and methods of using such crystalline form in mammals suffering from inflammatory bowel diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No.15/165,126, filed on May 26, 2016; which application claims the benefitof U.S. Provisional Application Nos. 62/167,694, filed on May 28, 2015,and 62/312,273, filed on Mar. 23, 2016, the disclosures of which areincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

Field of the Invention

The invention is directed to naphthyridine compounds useful as JAKkinase inhibitors. The invention is also directed to pharmaceuticalcompositions comprising such compounds, methods of using such compoundsto treat inflammatory diseases, and processes and intermediates usefulfor preparing such compounds.

State of the Art

Ulcerative colitis is a chronic inflammatory disease of the colon. Thedisease is characterized by inflammation and ulceration of the mucosallayer of the rectum and the large intestine. Common symptoms includediarrhea, bloody stools, and abdominal pain. The clinical course isintermittent, marked by alternating periods of exacerbation andremission. Incidence seems to be greater in developed than in developingcountries. An estimated 1.2 million people in major industrializedcountries suffer from ulcerative colitis and the numbers are expected toincrease along with population growth. Patients with ulcerative colitisare at an increased risk of developing colorectal cancer. (e.g. Daneseet al. N Engl J Med, 2011, 365, 1713-1725).

Although there exists a variety of therapeutic options to promote andmaintain remission of ulcerative colitis (UC) in patients, none isideal. Sulfasalazine-related treatments are often effective in mild UC,but much less so in moderate to severe disease. Corticosteroids areoften used to provide rapid induction of remission in patients withmoderate to severe UC. However, chronic use of steroids to maintainremission is discouraged due to their association with longer termadverse effects (e.g., osteoporosis and fractures, infections,cataracts, slower wound healing and suppression of adrenal gland hormoneproduction). Systemic immunosuppressants such as azathioprine,cyclosporine and methotrexate have a slow onset and modest efficacy inmoderate to severe UC patients, but prolonged use can be problematic dueto consequences of long-term systemic immunosuppression (e.g., increasedrisk of infections and lymphoma). Anti-TNFα antibodies (e.g., infliximaband adalimumab), while expensive and requiring subcutaneous orintravenous administration, are efficacious in approximately 60 to 70%of UC patients with moderate to severe disease. However, up to one thirdof patients fail to respond adequately, while another third of initialresponders develop tolerance over a few weeks (Allez et al., J Crohn'sColitis, 2010, 4, 355-366; Rutgeerts et al., N Engl J Med, 2005, 353,2462-2476). The most recently approved UC therapy, vedolizumab, ananti-α₄β₇ integrin antibody, is efficacious in moderate to severe UCpatients although its parenteral route is suboptimal, and theconsequences of long-term immunosuppression via this mechanism remain tobe determined. Despite existing therapeutic options, about 10 to 20% ofUC patients still require colectomy within 10 years of diagnosis(Targownik et al., Am J Gastroenterol, 2012, 107, 1228-1235). It isclear there remains an unmet medical need for an effective therapy topromote and maintain remission of moderate to severe UC without thesafety concerns resulting from chronic, systemic immunosuppression.

While the mechanism underlying ulcerative colitis is not completelyunderstood, it is believed that environmental factors in geneticallysusceptible individuals evoke an inappropriate (excessive) reaction bythe immune system to gut microbiota, resulting in colonic inflammation,tissue damage, and the associated symptoms characteristic of thedisease.

Although the precise pathogenesis of UC is unclear, it is apparent thatproinflammatory cytokines play a pivotal role in the immunologicalresponse (Strober et al., Gastroenterol, 2011, 140, 1756-1767). Many ofthe proinflammatory cytokines most commonly elevated in UC (e.g., IL-4,IL-6, IL-13, IL-15, IL-23, IL-24, IFNγ and leptin), rely on the JAKfamily of tyrosine kinases (i.e., JAK1, JAK2, JAK3 and Tyk2) for signaltransduction. Ligand binding to a cytokine receptor triggersautophosphorylation of its associated JAK, which in turn results inphosphorylation of a signal transducer and activator of transduction(STAT) protein. Different STATs form hetero- or homodimers and promotetranscription of their target genes in the cell nucleus to regulatefunctions such as cell growth, differentiation and death (Clark et al.,J Med Chem, 2014, 57, 5023-5038).

Inhibition of the family of JAK enzymes could inhibit signaling of manykey pro-inflammatory cytokines. Thus JAK inhibitors are likely to beuseful in the treatment of ulcerative colitis and other inflammatorydiseases such as Crohn's disease, allergic rhinitis, asthma, and chronicobstructive pulmonary disease (COPD). However, due to the modulatingeffect of the JAK/STAT pathway on the immune system, systemic exposureto JAK inhibitors may have an adverse systemic immunosuppressive effect.It would be desirable, therefore, to provide new JAK inhibitors whichhave their effect at the site of action without significant systemiceffects. In particular, for the treatment of gastrointestinalinflammatory diseases, such as ulcerative colitis, it would be desirableto provide new JAK inhibitors which can be administered orally andachieve therapeutically relevant exposure in the gastrointestinal tractwith minimal systemic exposure.

SUMMARY OF THE INVENTION

In one aspect, the invention provides novel compounds having activity asJAK kinase inhibitors.

-   -   Accordingly, the invention provides a compound of formula (I):

wherein

R¹ is selected from:

-   -   (a) C₁₋₄alkyl, wherein C₁₋₄alkyl is optionally substituted with        one, two, or three fluoro or with a substituent selected from        —CN; —OC₁₋₃alkyl; —C(O)OC₁₋₄alkyl; phenyl, wherein phenyl is        optionally substituted with —OH; pyridinyl, wherein pyridinyl is        optionally substituted with —CN; tetrahydropyranyl;        —C(O)NR^(a)R^(b), wherein R^(a) and R^(b) are independently        hydrogen or C₁₋₃alkyl or R^(a) is hydrogen and R^(b) is

-   -    and a group selected from

-   -   (b) a group selected from

-   -    wherein m is 1 or 2;    -   (c) —C(O)R⁶, wherein R⁶ is selected from        -   C₁₋₄alkyl, wherein C₁₋₄alkyl is optionally substituted with            one, two, or three fluoro or with a substituent selected            from —OH, —CN, —OC₁₋₄alkyl, phenyl, and —NR^(e)R^(f),            wherein R^(e) and R^(f) are independently hydrogen or            C₁₋₃alkyl;        -   C₃₋₆cycloalkyl, wherein C₃₋₆cycloalkyl is optionally            substituted with C₁₋₃alkyl;        -   pyridinyl, wherein pyridinyl is optionally substituted with            —CN; and

-   -   -    wherein R⁷ is —CN, —CF₃, or —OCH₃,

    -   (d) —C(O)OR⁸, wherein R⁸ is selected from        -   C₁₋₄alkyl, wherein C₁₋₄alkyl is optionally substituted with            —CN, C₃₋₆cycloalkyl, tetrahydrofuranyl, or —OR^(m), wherein            R^(m) is hydrogen or C₁₋₃alkyl; and        -   C₁₋₄alkenyl; and

    -   (e) —S(O)₂R⁹, wherein R⁹ is selected from        -   C₁₋₄alkyl, wherein C₁₋₄alkyl is optionally substituted with            —CN, —OC₁₋₃alkyl, phenyl, pyridinyl, or C₃₋₆cycloalkyl,        -   C₁₋₄alkenyl,        -   C₃₋₆cycloalkyl, wherein C₃₋₆cycloalkyl is optionally            substituted with C₁₋₃alkyl,        -   phenyl,        -   pyridinyl, wherein pyridinyl is optionally substituted with            fluoro,        -   heterocycle containing 4 to 6 ring atoms including one            nitrogen atom, wherein the heterocycle is optionally            substituted with —CN or C₁₋₃alkyl, wherein C₁₋₃alkyl is            optionally substituted with —CN or —OC₁₋₃alkyl; and

R² is selected from hydrogen, —OC₁₋₃alkyl, and —CH₂—R¹⁰, wherein R¹⁰ isselected from —OH, morpholinyl, piperidinyl, wherein piperidinyl isoptionally substituted with two fluoro, and piperazinyl, whereinpiperazinyl is optionally substituted with methyl;

R³ is selected from hydrogen, C₁₋₃alkyl, —OC₁₋₃alkyl, —C(O)OC₁₋₃alkyl,—S(O)₂C₁₋₃alkyl, and —CH₂S(O)₂C₁₋₃alkyl;

R⁴ is hydrogen or —OC₁₋₃alkyl;

R⁵ is hydrogen or fluoro; and

n is 1 or 2;

provided that

-   -   when R³ is —OC₁₋₃alkyl and R², R⁴, and R⁵ are each hydrogen, R⁹        is not phenyl;    -   when R⁵ is fluoro, n is 1, and R², R³, and R⁴ are each hydrogen,        R⁹ is not phenyl; and    -   when R⁵ is fluoro, R³ is methyl, and R² and R⁴ are each        hydrogen, R¹ is not —C(O)OR⁸;

or a pharmaceutically-acceptable salt or stereoisomer thereof.

As used hereinafter, the phrase “compound of formula (I)” means acompound of formula (I) or a pharmaceutically acceptable salt thereof;i.e., this phrase means a compound of formula (I) in free base form orin a pharmaceutically acceptable salt form unless otherwise indicated.

The invention also provides a pharmaceutical composition comprising acompound of the invention and a pharmaceutically-acceptable carrier.

In another aspect, the invention provides a particular compound offormula (I) in crystalline free base form. Crystalline3-((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)propanenitrilehas been found to have a melting temperature in the range of about 243°C. to about 253° C., typically between about 246° C. and about 250° C.,a decomposition onset at about 237° C., and to exhibit weight changes ofless than about 0.15% when exposed to a range of relative humiditybetween about 5% and about 90% at room temperature. In yet anotheraspect, the invention provides a crystalline solvate of3-((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)propanenitrile.

The invention also provides a method of treating gastrointestinalinflammatory disease, in particular, ulcerative colitis in a mammal, themethod comprising administering to the mammal a therapeuticallyeffective amount of a compound or of a pharmaceutical composition of theinvention. In separate and distinct aspects, the invention also providessynthetic processes and intermediates described herein, which are usefulfor preparing compounds of the invention.

The invention also provides a compound of the invention as describedherein for use in medical therapy, as well as the use of a compound ofthe invention in the manufacture of a formulation or medicament fortreating gastrointestinal inflammatory disease in a mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects of the present invention are illustrated by reference tothe accompanying drawings.

FIG. 1 shows a powder x-ray diffraction (PXRD) pattern of crystallineForm I3-((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)propanenitrile[hereinafter Form I].

FIG. 2 shows a differential scanning calorimetry (DSC) thermogram ofcrystalline Form I.

FIG. 3 shows a thermal gravimetric analysis (TGA) plot of crystallineForm I.

FIG. 4 shows a dynamic moisture sorption (DMS) isotherm of crystallineForm I observed at a temperature of about 25° C.

FIG. 5 shows a powder X-ray diffraction (PXRD) pattern of thecrystalline Form II solvate3-((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)propanenitrile.

DETAILED DESCRIPTION OF THE INVENTION

Among other aspects, the invention provides JAK kinase inhibitors offormula (I), pharmaceutically-acceptable salts thereof, andintermediates for the preparation thereof. The following substituentsand values are intended to provide representative examples of variousaspects of this invention. These representative values are intended tofurther define such aspects and are not intended to exclude other valuesor limit the scope of the invention.

In a specific aspect, R¹ is C₁₋₄alkyl, wherein C₁₋₄alkyl is optionallysubstituted with one, two, or three fluoro or with a substituentselected from —CN; —OC₁₋₃alkyl; —C(O)OC₁₋₄alkyl; phenyl, wherein phenylis optionally substituted with —OH; pyridinyl, wherein pyridinyl isoptionally substituted with —CN; tetrahydropyranyl; —C(O)NR^(a)R^(b),wherein R^(a) and R^(b) are independently hydrogen or C₁₋₃alkyl or R^(a)is hydrogen and R^(b) is

and a group selected from

In another specific aspect, R¹ is C₁₋₄alkyl, wherein C₁₋₄alkyl isoptionally substituted with one, two, or three fluoro or with asubstituent selected from —CN; —OC₁₋₃alkyl; phenyl, wherein phenyl isoptionally substituted with —OH; pyridinyl, wherein pyridinyl isoptionally substituted with —CN; tetrahydropyranyl; —C(O)NHCH₃; and

In another specific aspect, R¹ is C₁₋₄alkyl, wherein C₁₋₄alkyl issubstituted with one, two, or three fluoro, or with —CN, or —C(O)NHCH₃.

In specific aspects, R¹ is a group selected from

wherein m is 1 or 2, or wherein m is 1.

In a specific aspect, R¹ is —C(O)R⁶, wherein R⁶ is selected fromC₁₋₄alkyl, wherein C₁₋₄alkyl is optionally substituted with one, two, orthree fluoro or with a substituent selected from —OH, —CN, —OC₁₋₄alkyl,phenyl, and —NR^(e)R^(f), wherein R^(e) and R^(f) are independentlyhydrogen or C₁₋₃alkyl; C₃₋₆cycloalkyl, wherein C₃₋₆cycloalkyl isoptionally substituted with C₁₋₃alkyl; pyridinyl, wherein pyridinyl isoptionally substituted with —CN; and

wherein R⁷ is —CN, —CF₃, or —OCH₃.

In another specific aspect, R¹ is —C(O)R⁶, wherein R⁶ is selected fromC₁₋₄alkyl, wherein C₁₋₄alkyl is optionally substituted with one, two, orthree fluoro or with a substituent selected from —OH and phenyl;C₃₋₆cycloalkyl, wherein C₃₋₆cycloalkyl is optionally substituted withC₁₋₃alkyl; and

wherein R⁷ is —CN or —CF₃.

In another specific aspect, R¹ is —C(O)R⁶, wherein R⁶ is C₁₋₄alkyl,wherein C₁₋₄alkyl is substituted with one, two, or three fluoro, or with—CN or —C(O)NHCH₃.

In a specific aspect, R¹ is —C(O)OR⁸, wherein R⁸ is selected fromC₁₋₄alkyl, wherein C₁₋₄alkyl is optionally substituted with —CN,C₃₋₆cycloalkyl, tetrahydrofuranyl, or —OR^(m), wherein R^(m) is hydrogenor C₁₋₃alkyl; and C₁₋₄alkenyl.

In a specific aspect, R¹ is —S(O)₂R⁹, wherein R⁹ is selected fromC₁₋₄alkyl, wherein C₁₋₄alkyl is optionally substituted with —CN,—OC₁₋₃alkyl, phenyl, pyridinyl, or C₃₋₆cycloalkyl; C₃₋₆cycloalkyl,wherein C₃₋₆cycloalkyl is optionally substituted with C₁₋₃alkyl; phenyl;pyridinyl, wherein pyridinyl is optionally substituted with fluoro;heterocycle containing 4 to 6 ring atoms including one nitrogen atom,wherein the heterocycle is optionally substituted with —CN or C₁₋₃alkyl,wherein C₁₋₃alkyl is optionally substituted with —CN or —OC₁₋₃alkyl; and

In another specific aspect, R¹ is —S(O)₂R⁹, wherein R⁹ is selected fromwherein C₁₋₄alkyl is optionally substituted with —CN, —OC₁₋₃alkyl,phenyl, pyridinyl, or C₃₋₆cycloalkyl; C₃₋₆cycloalkyl, whereinC₃₋₆cycloalkyl is optionally substituted with C₁₋₃alkyl; pyridinyl,wherein pyridinyl is optionally substituted with fluoro; heterocyclecontaining 4 or 5 ring atoms including one nitrogen atom, wherein theheterocycle is bonded to sulfur through the nitrogen atom and theheterocycle is optionally substituted with —CN or with —CH₂OCH₃; and

In another specific aspect, R¹ is —S(O)₂R⁹, wherein R⁹ is pyridinyl.

In yet another aspect, R¹ is selected from —(CH₂)₂CN, —CH₂CH₂F,—CH₂C(O)NHCH₃, —C(O)CHF₂, and —S(O)₂-pyridin-3-yl.

In a specific aspect, R² is selected from hydrogen, —OC₁₋₃alkyl, and—CH₂—R¹⁰, wherein R¹⁰ is selected from —OH, morpholinyl, piperidinyl,wherein piperidinyl is optionally substituted with two fluoro, andpiperazinyl, wherein piperazinyl is optionally substituted with methyl.

In another specific aspect, R² is selected from hydrogen, —OCH₃, and—CH₂—R¹⁰, wherein R¹⁰ is selected from —OH, morpholinyl, piperidinyl,wherein piperidinyl is substituted with 2 fluoro at the 4-position, andpiperazinyl, wherein piperazinyl is substituted with methyl at the4-position.

In yet another specific aspect, R² is hydrogen.

In a specific aspect, R³ is selected from hydrogen, C₁₋₃alkyl,—OC₁₋₃alkyl, —C(O)OC₁₋₃alkyl, —S(O)₂C₁₋₃alkyl, and —CH₂S(O)₂C₁₋₃alkyl.

In another specific aspect, R³ is selected from hydrogen, —CH₃, —OCH₃,and —C(O)OCH₃.

In yet another specific aspect, R³ is hydrogen.

In specific aspects, R⁴ is hydrogen or —OC₁₋₃alkyl; or R⁴ is hydrogen or—OCH₃, or R⁴ is hydrogen.

In specific aspects, R⁵ is hydrogen or fluoro, or R⁵ is hydrogen.

In a specific aspect n is 1. In another specific aspect n is 2.

In a specific aspect, the invention provides a compound of formula (I)wherein:

R¹ is selected from:

(a) wherein C₁₋₄alkyl is optionally substituted with one, two, or threefluoro or with a substituent selected from —CN; —OC₁₋₃alkyl; phenyl,wherein phenyl is optionally substituted with —OH; pyridinyl, whereinpyridinyl is optionally substituted with —CN; tetrahydropyranyl;—C(O)NHCH₃; and

(b) a group selected from

wherein m is 1;

(c) —C(O)R⁶, wherein R⁶ is selected from C₁₋₄alkyl, wherein C₁₋₄alkyl isoptionally substituted with one, two, or three fluoro or with asubstituent selected from —OH and phenyl; C₃₋₆cycloalkyl, whereinC₃₋₆cycloalkyl is optionally substituted with C₁₋₃alkyl; and

wherein R⁷ is —CN or —CF₃;

(d) —C(O)OR⁸ wherein R⁸ is selected from C₁₋₄alkyl, wherein C₁₋₄alkyl isoptionally substituted with —CN, C₃₋₆cycloalkyl, tetrahydrofuranyl, or—OR^(m), wherein R^(m) is hydrogen or C₁₋₃alkyl; and C₁₋₄alkenyl; and

(e) —S(O)₂R⁹, wherein R⁹ is selected from C₁₋₄alkyl, wherein C₁₋₄alkylis optionally substituted with —CN, —OC₁₋₃alkyl, phenyl, pyridinyl, orC₃₋₆cycloalkyl; C₃₋₆cycloalkyl, wherein C₃₋₆cycloalkyl is optionallysubstituted with C₁₋₃alkyl; pyridinyl, wherein pyridinyl is optionallysubstituted with fluoro; heterocycle containing 4 or 5 ring atomsincluding one nitrogen atom, wherein the heterocycle is bonded to sulfurthrough the nitrogen atom and the heterocycle is optionally substitutedwith —CN or —CH₂OCH₃; and

R² is selected from hydrogen, —OCH₃, and —CH₂—R¹⁰, wherein R¹⁰ isselected from —OH, morpholinyl, piperidinyl, wherein piperidinyl issubstituted with two fluoro at the 4-position, and piperazinyl, whereinpiperazinyl is substituted with methyl at the 4-position;

R³ is selected from hydrogen, —CH₃, —OCH₃, and —C(O)OCH₃;

R⁴ is hydrogen or —OCH₃;

R⁵ is hydrogen or fluoro; and

n is 1 or 2,

provided that when R⁵ is fluoro, R³ is hydrogen.

In another specific aspect, the invention provides a compound of formula(I) wherein:

R¹ is selected from:

(a) C₁₋₄alkyl, wherein C₁₋₄alkyl is substituted with one, two, or threefluoro, or with —CN or —C(O)NHCH₃;

(c) —C(O)R⁶, wherein R⁶ is C₁₋₄alkyl, wherein C₁₋₄alkyl is substitutedwith one, two, or three fluoro; and

(e) —S(O)₂R⁹ wherein R⁹ is pyridinyl;

R², R³, R⁴, and R⁵ are each hydrogen; and

n is 1 or 2.

In a certain aspect, the invention provides compounds of formula (II):

wherein the variable R¹ is as defined herein.

In another aspect, the invention provides compounds of formula (III):

wherein the variable R¹ is as defined herein.

In one aspect, the invention provides the compounds of Examples 1-23 andTables 1-8 below

In another aspect, the invention provides a compound selected from thefollowing compounds

-   3-((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)propanenitrile,-   N⁵-((1R,3s,5S)-8-(2-fluoroethyl)-8-azabicyclo[3.2.1]octan-3-yl)-N⁷-(5-methyl-1H-pyrazol-3-yl)-1,6-naphthyridine-5,7-diamine,-   N⁷-(5-methyl-1H-pyrazol-3-yl)-N⁵-((1R,3s,5S)-8-(pyridin-3-ylsulfonyl)-8-azabicyclo[3.2.1]octan-3-yl)-1,6-naphthyridine-5,7-diamine,-   2-(dimethylamino)-1-((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)ethan-1-one,-   2,2-difluoro-1-((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)ethan-1-one,-   N⁵-((1R,3s,5S)-8-((2-methoxyethyl)sulfonyl)-8-azabicyclo[3.2.1]octan-3-yl)-N⁷-(5-methyl-1H-pyrazol-3-yl)-1,6-naphthyridine-5,7-diamine,-   N⁷-(5-methyl-1H-pyrazol-3-yl)-N⁵-((1R,3s,5S)-9-(pyridin-3-ylsulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)-1,6-naphthyridine-5,7-diamine,-   isobutyl    (1R,3s,5S)-3-((2-(hydroxymethyl)-7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylate,-   N-methyl-2-((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)acetamide,    and pharmaceutically acceptable salts thereof.

Chemical structures are named herein according to IUPAC conventions asimplemented in ChemDraw software (PerkinElmer, Inc., Cambridge, Mass.).For example, the compound of Example 1:

is designated as3-((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)propanenitrile.The (1R,3s,5S) notation describes the exo orientation of thenaphthyridinylamino group with respect to the 8-azabicyclo-[3.2.1]octanegroup. All of the compounds of the invention are in the exo orientation.

Furthermore, the pyrazolyl moiety of the compounds of formula (I) existsin tautomeric form. For example, the compound of Example 1 mayequivalently be represented as:

According to the IUPAC convention, these representations give rise todifferent numbering of the atoms of the pyrazolyl portion. The aboverepresentation is designated as3-((1R,3s,5S)-3-((7-((3-methyl-1H-pyrazol-5-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)propanenitrile,where the underlining identifies where the name differs from that of thefirst representation. It will be understood that although structures areshown, or named, in a particular form, the invention also includes thetautomer thereof.

The compounds of the invention contain one or more chiral centers andtherefore, such compounds (and intermediates thereof) can exist asracemic mixtures; pure stereoisomers (i.e., enantiomers ordiastereomers); stereoisomer-enriched mixtures and the like. Chiralcompounds shown or named herein without a defined stereochemistry at achiral center are intended to include any or all possible stereoisomervariations at the undefined stereocenter unless otherwise indicated. Thedepiction or naming of a particular stereoisomer means the indicatedstereocenter has the designated stereochemistry with the understandingthat minor amounts of other stereoisomers may also be present unlessotherwise indicated, provided that the utility of the depicted or namedcompound is not eliminated by the presence of another stereoisomer.

Compounds of formula (I) also contain several basic groups (e.g., aminogroups) and therefore, such compounds can exist as the free base or invarious salt forms, such a mono-protonated salt form, a di-protonatedsalt form, a tri-protonated salt form, or mixtures thereof. All suchforms are included within the scope of this invention, unless otherwiseindicated.

This invention also includes isotopically-labeled compounds of formula(I), i.e., compounds of formula (I) where an atom has been replaced orenriched with an atom having the same atomic number but an atomic massdifferent from the atomic mass that predominates in nature. Examples ofisotopes that may be incorporated into a compound of formula (I)include, but are not limited to, ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O,¹⁷O, ¹⁸O, ³⁵S, ³⁶Cl, and ¹⁸F. Of particular interest are compounds offormula (I) enriched in tritium or carbon-14, which compounds can beused, for example, in tissue distribution studies. Also of particularinterest are compounds of formula (I) enriched in deuterium especiallyat a site of metabolism, which compounds are expected to have greatermetabolic stability. Additionally of particular interest are compoundsof formula (I) enriched in a positron emitting isotope, such as ¹¹C,¹⁸F, ¹⁵O and ¹³N, which compounds can be used, for example, in PositronEmission Tomography (PET) studies.

Definitions

When describing this invention including its various aspects andembodiments, the following terms have the following meanings, unlessotherwise indicated.

The term “alkyl” means a monovalent saturated hydrocarbon group whichmay be linear or branched or combinations thereof. Unless otherwisedefined, such alkyl groups typically contain from 1 to 10 carbon atoms.Representative alkyl groups include, by way of example, methyl (Me),ethyl (Et), n-propyl (n-Pr) or (nPr), isopropyl (i-Pr) or (iPr), n-butyl(n-Bu) or (nBu), sec-butyl, isobutyl, tert-butyl (t-Bu) or (tBu),n-pentyl, n-hexyl, 2,2-dimethylpropyl, 2-methylbutyl, 3-methylbutyl,2-ethylbutyl, 2,2-dimethylpentyl, 2-propylpentyl, and the like

When a specific number of carbon atoms are intended for a particularterm, the number of carbon atoms is shown preceding the term. Forexample, the term “C₁₋₃ alkyl” means an alkyl group having from 1 to 3carbon atoms wherein the carbon atoms are in any chemically-acceptableconfiguration, including linear or branched configurations.

The term “alkoxy” means the monovalent group —O-alkyl, where alkyl isdefined as above. Representative alkoxy groups include, by way ofexample, methoxy, ethoxy, propoxy, butoxy, and the like.

The term “cycloalkyl” means a monovalent saturated carbocyclic groupwhich may be monocyclic or multicyclic. Unless otherwise defined, suchcycloalkyl groups typically contain from 3 to 10 carbon atoms.Representative cycloalkyl groups include, by way of example, cyclopropyl(cPr), cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl,adamantyl, and the like.

The term “heterocycle”, “heterocyclic”, or “heterocyclic ring” means amonovalent saturated or partially unsaturated cyclic non-aromatic group,having from 3 to 10 total ring atoms, wherein the ring contains from 2to 9 carbon ring atoms and from 1 to 4 ring heteroatoms selected fromnitrogen, oxygen, and sulfur. Heterocyclic groups may be monocyclic ormulticyclic (i.e., fused or bridged). Representative heterocyclic groupsinclude, by way of example, pyrrolidinyl, piperidinyl, piperazinyl,imidazolidinyl, morpholinyl, thiomorpholyl, indolin-3-yl,2-imidazolinyl, tetrahydropyranyl, 1,2,3,4-tetrahydroisoquinolin-2-yl,quinuclidinyl, 7-azanorbornanyl, nortropanyl, and the like, where thepoint of attachment is at any available carbon or nitrogen ring atom.Where the context makes the point of attachment of the heterocyclicgroup evident, such groups may alternatively be referred to as anon-valent species, i.e. pyrrolidine, piperidine, piperazine, imidazole,tetrahydropyran etc.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need of treatment.

The term “treatment” as used herein means the treatment of a disease,disorder, or medical condition (such as a gastrointestinal inflammatorydisease), in a patient, such as a mammal (particularly a human) whichincludes one or more of the following:

(a) preventing the disease, disorder, or medical condition fromoccurring, i.e., preventing the reoccurrence of the disease or medicalcondition or prophylactic treatment of a patient that is pre-disposed tothe disease or medical condition;

(b) ameliorating the disease, disorder, or medical condition, i.e.,eliminating or causing regression of the disease, disorder, or medicalcondition in a patient, including counteracting the effects of othertherapeutic agents;

(c) suppressing the disease, disorder, or medical condition, i.e.,slowing or arresting the development of the disease, disorder, ormedical condition in a patient; or

(d) alleviating the symptoms of the disease, disorder, or medicalcondition in a patient.

The term “pharmaceutically acceptable salt” means a salt that isacceptable for administration to a patient or a mammal, such as a human(e.g., salts having acceptable mammalian safety for a given dosageregime). Representative pharmaceutically acceptable salts include saltsof acetic, ascorbic, benzenesulfonic, benzoic, camphorsulfonic, citric,ethanesulfonic, edisylic, fumaric, gentisic, gluconic, glucoronic,glutamic, hippuric, hydrobromic, hydrochloric, isethionic, lactic,lactobionic, maleic, malic, mandelic, methanesulfonic, mucic,naphthalenesulfonic, naphthalene-1,5-disulfonic,naphthalene-2,6-disulfonic, nicotinic, nitric, orotic, pamoic,pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonicand xinafoic acid, and the like.

The term “salt thereof” means a compound formed when the hydrogen of anacid is replaced by a cation, such as a metal cation or an organiccation and the like. For example, the cation can be a protonated form ofa compound of formula (I), i.e. a form where one or more amino groupshave been protonated by an acid. Typically, the salt is apharmaceutically acceptable salt, although this is not required forsalts of intermediate compounds that are not intended for administrationto a patient.

The term “amino-protecting group” means a protecting group suitable forpreventing undesired reactions at an amino nitrogen. Representativeamino-protecting groups include, but are not limited to, formyl; acylgroups, for example alkanoyl groups, such as acetyl andtri-fluoroacetyl; alkoxycarbonyl groups, such as tent butoxycarbonyl(Boc); arylmethoxycarbonyl groups, such as benzyloxycarbonyl (Cbz) and9-fluorenylmethoxycarbonyl (Fmoc); arylmethyl groups, such as benzyl(Bn), trityl (Tr), and 1,1-di-(4′-methoxyphenyl)methyl; silyl groups,such as trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS),[2-(trimethylsilyl)ethoxy]methyl (SEM); and the like. Numerousprotecting groups, and their introduction and removal, are described inT. W. Greene and G. M. Wuts, Protecting Groups in Organic Synthesis,Third Edition, Wiley, N.Y.

General Synthetic Procedures

Compounds of this invention, and intermediates thereof, can be preparedaccording to the following general methods and procedures usingcommercially-available or routinely-prepared starting materials andreagents. The substituents and variables (e.g., R¹, R², R³, R⁴, etc.)used in the following schemes have the same meanings as those definedelsewhere herein unless otherwise indicated. Additionally, compoundshaving an acidic or basic atom or functional group may be used or may beproduced as a salt unless otherwise indicated (in some cases, the use ofa salt in a particular reaction will require conversion of the salt to anon-salt form, e.g., a free base, using routine procedures beforeconducting the reaction).

Although a particular embodiment of the present invention may be shownor described in the following procedures, those skilled in the art willrecognize that other embodiments or aspects of the present invention canalso be prepared using such procedures or by using other methods,reagents, and starting materials know to those skilled in the art. Inparticular, it will be appreciated that compounds of the invention maybe prepared by a variety of process routes in which reactants arecombined in different orders to provide different intermediates en routeto producing final products.

A general method of preparing final compounds of the invention utilizesa key intermediate 1 as illustrated in Scheme 1. The variables R¹, R²,R³, R⁴, R⁵, R⁶, R⁸, and R⁹ are defined as in formula (I), R^(A)represents an optionally substituted C₁₋₄alkyl, and L is a leavinggroup. For simplicity, the scheme shows compounds in which the variablen of formula (I) is defined as 1, i.e. the bicyclo group is8-azabicyclo[3.2.1]octyl. An analogous process is used to preparecompounds in which the variable n is 2.

To prepare compounds in which R¹ is an optionally substituted alkylgroup as defined in option (a), the alkylation reaction typically uses ahalo leaving group L, principally bromo or iodo, although alternativeleaving groups such as hydroxy or trifluoromethanesulfonate (commonlytriflate) may also be employed. The reaction is typically conducted bycontacting intermediate 1 with an excess of the reagent L-R^(A) in aninert diluent in the presence of an excess of base. The reaction istypically conducted at a temperature between about 20 and about 60° C.for between about 10 and 24 hours or until the reaction is substantiallycomplete.

Alternatively, the Michael addition reaction may be used to preparecompounds in which R¹ is a cyanoethyl group. For example, as describedin the examples below, to prepare a compound in which R¹ is —(CH₂)₂CN,intermediate 1 is contacted with an excess, for example 1.1 to 1.5equivalents, of acrylonitrile in the presence of an excess of base, forexample diisopropylethylamine or diazobicycloundecene. The reaction istypically conducted at room temperature for between about 3 and about 24hours or until the reaction is substantially complete. In certain casesit is useful to prepare compounds in which R¹ is an optionallysubstituted alkyl group by reductive amination with a suitably selectedaldehyde.

Compounds in which R¹ is defined as —C(O)R⁶ may be prepared bycontacting intermediate 1 with a modest excess of carboxylic acidreagent HO—C(O)—R⁶ under typical amide coupling conditions. The reactionis typically performed in the presence of an excess of base utilizing anactivating agent such asN,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate (HATU). The reaction is typically conducted at roomtemperature for between about 3 and about 24 hours or until the reactionis substantially complete.

A chloroformate reagent Cl—C(O)OR⁸ may be used to prepare carbamatecompounds in which R¹ is defined as —C(O)OR⁸. Typically, intermediate 1is contacted with about 1 equivalent of the chloroformate in thepresence of an excess of base at a temperature on the order of 0° C. Thereaction is typically conducted for between about 1 and about 3 hours oruntil the reaction is substantially complete.

Sulfonamide compounds in which R¹ is defined as —S(O)₂R⁹ are typicallyprepared by contacting intermediate 1 with between about 1 and about 1.1equivalents of a sulfonylchloride of the form Cl—S(O)₂R⁹ in the presenceof an excess of base at a temperature on the order of 0° C. The reactionis typically conducted for between about 1 and about 24 hours or untilthe reaction is substantially complete.

An exemplary reaction for the preparation of intermediate 1-2 in whichthe variables R², R³, R⁴, and R⁵ are each hydrogen is illustrated inScheme 2.

In the amine displacement reaction of step 1, thedi-chloro-naphthyridine 2 is reacted with a slight excess of atert-butoxycarbonyl (Boc)-protected amino-8-azabicyclo-[3.2.1]octane 3in the presence of base to provide intermediate 4. The Boc-protectedintermediate amino-methyl-pyrazole 5 is then reacted with intermediate 4under standard Buchwald amination conditions. For example intermediate 4is combined with between about 1 and about 1.5 equivalents of thepyrazole intermediate 5 in the presence of a base, such as cesiumcarbonate, and a palladium catalyst. The reaction is typically conductedat elevated temperature, between about 85° C. and about 110° C. forbetween about 24 and about 48 hours or until the reaction issubstantially complete. The Boc protecting group may be removed from themethylpyrazole during the course of the Buchwald reaction, as shown inScheme 2, or the Boc protecting group may remain on the methylpyrazoleand be removed along with the protecting group on the 8-azabicylooctylgroup in the final step. In the last step, the Boc group or groups maybe removed by standard treatment with an acid, typically trifluoroaceticacid or hydrochloric acid in dioxane.

As described in the appended examples, the preparation of intermediate 1in which one or more of R², R³, R⁴, and R⁵ is other than hydrogen mayfollow the sequence of steps shown in Scheme 2, starting with asubstituted di-chloro-naphthyridine reagent analogous to intermediate 2.The substituted intermediate 2 may be commercially available or easilyprepared from commercial reagents by standard procedures.

It will be appreciated that compounds of the invention may be preparedby a variety of process routes in which reactants are combined indifferent orders to provide different intermediates en route toproducing final products. For certain substituents, R², R³, R⁴, or R⁵ itis preferable to start with a naphthyridine intermediate substitutedwith a precursor of the desired substituent, perform the aminedisplacement reaction to add the protected amino-8-azabicylooctyl group,and transform the precursor to the desired substituent or one step awayfrom a desired substituent before adding the pyrazole group. Onespecific example, the process to form intermediate 6-3, theBoc-protected precursor to substituted intermediate 2 where R² is —CH₂OHand R³, R⁴, and R⁵ are each hydrogen, is sketched in Scheme 3 below anddescribed explicitly in Preparations 5 and 6.

The protected amino-8-azabicylooctyl group 3 is added to a hydroxylsubstituted di-chloro-naphthyridine 2-3 to form intermediate 7-3. Withthe 8-azabicylooctyl group installed, the hydroxyl substituent issuccessively transformed to the triflate, 8-3, and then to the methylester, 9-3, before adding the protected aminopyrazole 5 to formintermediate 10-3, which is hydrogenated to form the protectedintermediate 6-3 bearing the R² substituent —CH₂OH.

Additional synthetic processes for preparation of compounds of theinvention in which the substituents R², R³, R⁴ or R⁵ are other thanhydrogen and processes for the preparation of compounds in which thevariable n is 2, are described in the examples below.

Accordingly, in a method aspect, the invention provides a method ofpreparing a compound of formula (I) or a pharmaceutically acceptablesalt thereof, the method comprising reacting a compound of formula (IV):

with (i) a compound of formula L-R^(A) wherein L is a leaving group andR^(A) is an optionally substituted alkyl as defined for R¹ option (a) ora substituent of option (b), or (ii) HO—C(O)R⁶, or (iii) Cl—C(O)OR⁸, or(iv) Cl—S(O)₂R⁹ to form a compound of formula (I) or a pharmaceuticallyacceptable salt thereof.

In separate and distinct aspects, the invention provides a compound offormula (IV) wherein the variables take any of the values describedabove and a compound of formula (IV) wherein R², R³, R⁴, and R⁵ are eachhydrogen.

Crystalline Forms

In another aspect, the invention provides3-((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)propanenitrile(Example 1) in crystalline freebase form or a solvate thereof.

Crystalline Form I of the invention is a crystalline freebase of thecompound of Example 1. In one aspect, Form I is characterized by apowder X-ray diffraction (PXRD) pattern having significant diffractionpeaks, among other peaks, at 2θ values of 7.87±0.20, 12.78±0.20,15.78±0.20, and 20.41±0.20. Form I may be further characterized by aPXRD pattern having two or more additional diffraction peaks, includingthree or more and four or more additional diffraction peaks at 2θ valuesselected from 10.80±0.20, 13.47±0.20, 13.64±0.20, 14.66±0.20,15.11±0.20, 15.54±0.20, 17.75±0.20, 21.00±0.20, 22.22±0.20, 22.93±0.20,and 23.65±0.20. In another aspect, Form I is characterized by a PXRDpattern having diffraction peaks at 2θ values of 7.87±0.20, 10.80±0.20,12.78±0.20, 13.47±0.20, 13.64±0.20, 14.66±0.20, 15.11±0.20, 15.54±0.20,15.78±0.20, 17.75±0.20, 20.41±0.20, 21.00±0.20, 22.22±0.20, 22.93±0.20,and 23.65±0.20.

As is well known in the field of powder X-ray diffraction, peakpositions of PXRD spectra are relatively less sensitive to experimentaldetails, such as details of sample preparation and instrument geometry,than are the relative peak heights. Thus, in one aspect, the crystallineForm I is characterized by a powder x-ray diffraction pattern in whichthe peak positions are substantially in accordance with those shown inFIG. 1.

The structure of crystalline Form I has been further characterized bysingle crystal x-ray diffraction analysis. The crystals belong to amonoclinic crystal system and P2₁/n space group. The unit celldimensions are: a=8.8240(10) Å, b=22.4866(3) Å, c=10.2464(2) Å, α=90°,β=93.2360(10)°, γ=90°, volume=2029.87(5) Å³. The calculated density is1.317 g/cm³. The crystals contain four molecules per unit cell. Thestructure confirms that the crystals do not contain water or othersolvent molecules and the molecular structure is consistent with thestructure of the compound of Example 1 as depicted herein. Powder X-raydiffraction peaks predicted from the derived atomic positions are inexcellent agreement with observed results.

In another aspect, crystalline Form I is characterized by its behaviorwhen exposed to high temperature. As demonstrated in FIG. 2, thedifferential scanning calorimetry (DSC) trace recorded at a heating rateof 10° C. per minute exhibits a peak in endothermic heat flow,identified as a melt transition, in the range of about 243° C. to about253° C., including between about 246° C. and about 250° C. The thermalgravimetric analysis (TGA) trace of FIG. 3 shows no significant weightloss at temperatures below the onset of decomposition at about 237° C.

Crystalline Form I has been demonstrated to have a reversiblesorption/desorption profile with an exceptionally small propensity forhygroscopicity. Form I demonstrated less than about 0.14% weight gain inthe humidity range of 5% to 90% relative humidity and less than about0.07% weight gain in the humidity range of 5% to 90% relative humidityat room temperature, as shown in FIG. 4. No hysteresis was observed intwo cycles of sorption and desorption. Form I is considered to benon-hygroscopic.

Crystalline Form I has been shown to be stable upon upon exposure toelevated temperature and humidity. After 3 months at acceleratedconditions of 40° C. and 75% relative humidity, no statisticallysignificant changes in chemical content nor in impurity profile wereobserved.

Crystalline Form II is a crystalline solvate of the compound of Example1, characterized by the powder x-ray diffraction pattern of FIG. 5 withsignificant diffraction peaks, among other peaks, at 2θ values of9.76±0.20,15.06±0.20, 16.61±0.20, 20.40±0.20, and 21.99±0.20. Asdescribed in Example 18 below, the Form II solvate contains betweenabout 6% and about 7% methanol, between about 2% and about 2.5%N,N-dimethylformamide, and between about 1 and about 1.5% water.

The solvate of Form II may be prepared by adding a mixture of methanoland water, typically a ratio of methanol to water between about 1.5:1and about 3:1, to the product of the reaction ofN⁵-((1R,3s,5S)-8-azabicyclo[3.2.1]octan-3-yl)-N⁷-(5-methyl-1H-pyrazol-3-yl)-1,6-naphthyridine-5,7-diamine(compound 1-2 in Scheme 2) with acrylonitrile or withbromopropionitrile, which is typically conducted in dimethylformamide.The resulting reaction mixture is stirred at a temperature of betweenabout 20° C. and about 25° C. for between about 4 hours and about 24hours, filtered, and washed with methanol or a mixture of methanol andwater, such as a 1:1, 2:1, or 3:1 mixture of methanol and water toprovide the Form II solvate. An ethanol solvate may be prepared byslurrying the Form II solvate in ethanol.

The non-solvated crystalline Form I is conveniently prepared from asolvate form of the compound of Example 1, preferably the solvate ofForm II. In a typical process, the Form II solvate is typically combinedwith a non-protonating solvent to provide a slurry. Useful solventsinclude, but are not limited, to dioxane, toluene, butyl acetate, andacetone. The slurry is typically formed at a concentration of betweenabout 50 mg solvate to milliliter of solvent and about 85 mg/mL. Theslurry may be heated to a temperature of between about 40° C. and about110° C. for between about 4 hours and about 3 days, filtered, and washedto provide the Form I crystalline solid. As described in Examples 19 and20 below, acetone has been found to be a particularly useful solvent.

In another aspect, the invention provides a method of preparingcrystalline Form I, the method comprising (a) forming a slurry of acrystalline solvate of3-((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)propanenitrilein a solvent selected from dioxane, toluene, butyl acetate, and acetone,(b) heating the slurry at a temperature between about 40° C. and about110° C. for between about 4 hours and about 3 days, and (c) isolatingcrystalline Form I from the slurry.

Pharmaceutical Compositions

The compounds of the invention and pharmaceutically-acceptable saltsthereof are typically used in the form of a pharmaceutical compositionor formulation. Such pharmaceutical compositions may be administered toa patient by any acceptable route of administration including, but notlimited to, oral, rectal, nasal, inhaled, topical (includingtransdermal) and parenteral modes of administration.

Accordingly, in one of its compositions aspects, the invention isdirected to a pharmaceutical composition comprising apharmaceutically-acceptable carrier or excipient and a compound offormula (I), where, as defined above, “compound of formula (I)” means acompound of formula (I) or a pharmaceutically-acceptable salt thereof.Optionally, such pharmaceutical compositions may contain othertherapeutic and/or formulating agents if desired. When discussingcompositions and uses thereof, the “compound of the invention” may alsobe referred to herein as the “active agent”. As used herein, the term“compound of the invention” is intended to include all compoundsencompassed by formula (I) as well as the species embodied in formulas(II) and (III) and pharmaceutically-acceptable salts thereof

The pharmaceutical compositions of the invention typically contain atherapeutically effective amount of a compound of the present invention.Those skilled in the art will recognize, however, that a pharmaceuticalcomposition may contain more than a therapeutically effective amount,i.e., bulk compositions, or less than a therapeutically effectiveamount, i.e., individual unit doses designed for multiple administrationto achieve a therapeutically effective amount.

Typically, such pharmaceutical compositions will contain from about 0.1to about 95% by weight of the active agent; preferably, from about 5 toabout 70% by weight; and more preferably from about 10 to about 60% byweight of the active agent.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of the invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable pharmaceutical composition for a particular mode ofadministration is well within the scope of those skilled in thepharmaceutical arts. Additionally, the carriers or excipients used inthe pharmaceutical compositions of this invention arecommercially-available. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20th Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7th Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following:sugars, such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose, such as microcrystalline cellulose,and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin;talc; excipients, such as cocoa butter and suppository waxes; oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; glycols, such as propylene glycol; polyols,such as glycerin, sorbitol, mannitol and polyethylene glycol; esters,such as ethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; and other non-toxic compatible substances employed inpharmaceutical compositions.

Pharmaceutical compositions are typically prepared by thoroughly andintimately mixing or blending the active agent with apharmaceutically-acceptable carrier and one or more optionalingredients. The resulting uniformly blended mixture can then be shapedor loaded into tablets, capsules, pills and the like using conventionalprocedures and equipment.

The pharmaceutical compositions of the invention are preferably packagedin a unit dosage form. The term “unit dosage form” refers to aphysically discrete unit suitable for dosing a patient, i.e., each unitcontaining a predetermined quantity of active agent calculated toproduce the desired therapeutic effect either alone or in combinationwith one or more additional units. For example, such unit dosage formsmay be capsules, tablets, pills, and the like, or unit packages suitablefor parenteral administration.

In one embodiment, the pharmaceutical compositions of the invention aresuitable for oral administration. Suitable pharmaceutical compositionsfor oral administration may be in the form of capsules, tablets, pills,lozenges, cachets, dragees, powders, granules; or as a solution or asuspension in an aqueous or non-aqueous liquid; or as an oil-in-water orwater-in-oil liquid emulsion; or as an elixir or syrup; and the like;each containing a predetermined amount of a compound of the presentinvention as an active ingredient.

When intended for oral administration in a solid dosage form (i.e., ascapsules, tablets, pills and the like), the pharmaceutical compositionsof the invention will typically comprise the active agent and one ormore pharmaceutically-acceptable carriers, such as sodium citrate ordicalcium phosphate. Optionally or alternatively, such solid dosageforms may also comprise: fillers or extenders, such as starches,microcrystalline cellulose, lactose, dicalcium phosphate, sucrose,glucose, mannitol, and/or silicic acid; binders, such ascarboxymethylcellulose, hydroxypropylmethyl cellulose, alginates,gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, suchas glycerol; disintegrating agents, such as crosscarmellose sodium,crospovidone, agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and/or sodium carbonate; solutionretarding agents, such as paraffin; absorption accelerators, such asquaternary ammonium compounds; wetting agents, such as cetyl alcoholand/or glycerol monostearate; absorbents, such as kaolin and/orbentonite clay; lubricants, such as talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, and/ormixtures thereof; coloring agents; and buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants can also be presentin the pharmaceutical compositions of the invention. Examples ofpharmaceutically-acceptable antioxidants include: water-solubleantioxidants, such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfate, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole, butylated hydroxytoluene, lecithin, propyl gallate,alpha-tocopherol, and the like; and metal-chelating agents, such ascitric acid, ethylenediamine tetraacetic acid, sorbitol, tartaric acid,phosphoric acid, and the like. Coating agents for tablets, capsules,pills and like, include those used for enteric coatings, such ascellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropylmethylcellulose phthalate, methacrylic acid, methacrylic acid estercopolymers, cellulose acetate trimellitate, carboxymethyl ethylcellulose, hydroxypropyl methyl cellulose acetate succinate, and thelike.

Pharmaceutical compositions of the invention may also be formulated toprovide slow or controlled release of the active agent using, by way ofexample, hydroxypropyl methyl cellulose in varying proportions; or otherpolymer matrices, liposomes and/or microspheres. In addition, thepharmaceutical compositions of the invention may optionally containopacifying agents and may be formulated so that they release the activeingredient only, or preferentially, in a certain portion of thegastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active agent can also be in micro-encapsulated form, ifappropriate, with one or more of the above-described excipients.

Suitable liquid dosage forms for oral administration include, by way ofillustration, pharmaceutically-acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. Liquid dosage formstypically comprise the active agent and an inert diluent, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (esp., cottonseed, groundnut, corn, germ, olive, castor andsesame oils), oleic acid, glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof. Alternatively, certain liquid formulations can be converted,for example, by spray drying, to a powder, which is used to preparesolid dosage forms by conventional procedures.

Suspensions, in addition to the active ingredient, may containsuspending agents such as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

The compounds of this invention can also be administered parenterally(e.g. by intravenous, subcutaneous, intramuscular or intraperitonealinjection). For parenteral administration, the active agent is typicallyadmixed with a suitable vehicle for parenteral administration including,by way of example, sterile aqueous solutions, saline, low molecularweight alcohols such as propylene glycol, polyethylene glycol, vegetableoils, gelatin, fatty acid esters such as ethyl oleate, and the like.Parenteral formulations may also contain one or more anti-oxidants,solubilizers, stabilizers, preservatives, wetting agents, emulsifiers,buffering agents, or dispersing agents. These formulations may berendered sterile by use of a sterile injectable medium, a sterilizingagent, filtration, irradiation, or heat.

Alternatively, the pharmaceutical compositions of the invention areformulated for administration by inhalation. Suitable pharmaceuticalcompositions for administration by inhalation will typically be in theform of an aerosol or a powder. Such compositions are generallyadministered using well-known delivery devices, such as a metered-doseinhaler, a dry powder inhaler, a nebulizer or a similar delivery device.

When administered by inhalation using a pressurized container, thepharmaceutical compositions of the invention will typically comprise theactive ingredient and a suitable propellant, such asdichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas.Additionally, the pharmaceutical composition may be in the form of acapsule or cartridge (made, for example, from gelatin) comprising acompound of the invention and a powder suitable for use in a powderinhaler. Suitable powder bases include, by way of example, lactose orstarch.

The compounds of the invention can also be administered transdermallyusing known transdermal delivery systems and excipients. For example,the active agent can be admixed with permeation enhancers, such aspropylene glycol, polyethylene glycol monolaurate, azacycloalkan-2-onesand the like, and incorporated into a patch or similar delivery system.Additional excipients including gelling agents, emulsifiers and buffers,may be used in such transdermal compositions if desired.

Alternatively, compounds of the invention may be administered in theform of suppositories. A typical suppository formulation will generallyconsist of active agent with a binding and/or lubricating agent such asa gelatin or cocoa butter or other low melting vegetable or syntheticwax or fat.

The following non-limiting examples illustrate representativepharmaceutical compositions of the present invention.

Tablet Oral Solid Dosage Form

A compound of the invention or a pharmaceutically-acceptable saltthereof is dry blended with microcrystalline cellulose, polyvinylpyrrolidone, and croscarmellose sodium in a ratio of 4:5:1:1 andcompressed into tablets to provide a unit dosage of, for example, 5 mg,20 mg or 40 mg active agent per tablet.

Capsule Oral Solid Dosage Form

A compound of the invention or a pharmaceutically-acceptable saltthereof is combined with microcrystalline cellulose, polyvinylpyrrolidone, and crosscarmellose sodium in a ratio of 4:5:1:1 by wetgranulation and loaded into gelatin or hydroxypropyl methylcellulosecapsules to provide a unit dosage of, for example, 5 mg, 20 mg or 40 mgactive agent per capsule.

Tablet Oral Solid Dosage Form

A compound of the invention or a pharmaceutically-acceptable saltthereof is dry or wet granulated with microcrystalline cellulose,lactose, hydroxypropylmethyl cellulose, crospovidone, and magnesiumstearate. The formulation composition in % wt/wt is compound of theinvention (4%), microcrystalline cellulose (45%), lactose (36%),hydroxypropylmethyl cellulose (10%), crospovidone (3%), and magnesiumstearate (2%). The dry or wet granulated blends are compressed intotablets to provide a unit dosage of 10 mg active agent per 250 mgtablet.

Tablet Oral Solid Dosage Form

A compound of the invention or a pharmaceutically-acceptable saltthereof is dry or wet granulated with microcrystalline cellulose,hydroxypropylmethyl cellulose, crospovidone, and magnesium stearate. Theformulation composition in % wt/wt is compound of the invention (40%),microcrystalline cellulose (45%), hydroxypropylmethyl cellulose (10%),crospovidone (3%), and magnesium stearate (2%). The dry or wetgranulated blends are compressed into tablets to provide a unit dosageof 100 mg active agent per 250 mg tablet.

Liquid Formulation

A liquid formulation comprising a compound of the invention (0.1%),water (98.9%) and ascorbic acid (1.0%) is formed by adding a compound ofthe invention to a mixture of water and ascorbic acid.

Enteric Coated Oral Dosage Form

A compound of the invention is dissolved in an aqueous solutioncontaining polyvinyl pyrrolidone and spray coated onto microcrystallinecellulose or sugar beads in a ratio of 1:5 w/w active agent:beads andthen an approximately 5% weight gain of an enteric coating comprising anacrylic copolymer, for example a combination of acrylic copolymersavailable under the trade names Eudragit-L® and Eudragit-S®, orhydroxypropyl methylcellulose acetate succinate is applied. The entericcoated beads are loaded into gelatin or hydroxypropyl methylcellulosecapsules to provide a unit dosage of, for example, 30 mg active agentper capsule.

Enteric Coated Oral Dosage Form

An enteric coating comprising a combination of Eudragit-L® andEudragit-S®, or hydroxypropyl methylcellulose acetate succinate isapplied to a tablet oral dosage form or a capsule oral dosage formdescribed above.

Utility

The compounds of the invention have been shown to be potent inhibitorsof the JAK family of enzymes: JAK1, JAK2, JAK3, and TYK2. Inhibition ofthe family of JAK enzymes could inhibit signaling of many keypro-inflammatory cytokines. Thus the JAK inhibitors of the invention areexpected to be useful in the treatment of inflammatory diseases such asulcerative colitis, Crohn's disease, allergic rhinitis, asthma, andchronic obstructive pulmonary disease (COPD).

Compounds of the invention are designed to be poorly absorbed tominimize systemic exposure. As described in the experimental sectionbelow, the absorption and distribution of typical compounds has beenextensively profiled in preclinical assays. Selected compounds tested incannulated rats showed low absorption into plasma at the portal vein. Inaddition, the compounds are designed to have their effect at the site ofaction in the gastrointestinal tract. Selected compounds exhibited aratio of exposure in the colon to exposure in plasma in rat greater thanabout 450. In particular, the compound of Example 1 has demonstratedsignificantly higher exposure throughout the gastrointestinal tract thanexposure in plasma upon oral dosing in preclinical species. Furthermore,the compound of Example 1 has been evaluated in healthy human subjectsand was found to exhibit high drug concentration in stool samplessuggesting significant exposure in the gastrointestinal tract.

Oxazolone-induced colitis is an experimental model that has ahistological resemblance to human ulcerative colitis. As describedbelow, the compound of Example 1, among other compounds of theinvention, demonstrated activity in the oxazolone-induced colitis modelin mice. Further, when tested in an immunosuppression model in mice,which probes systemic functional activity, the compound demonstratedminimal effect of immunosuppression at the same dose required todemonstrate efficacy in the oxazolone model. Thus the compounddemonstrated anti-colitic activity without exhibiting systemic effectsin preclinical models.

It is expected that the high colon to plasma ratio achieved with thesecompounds will provide robust, luminally-driven anti-inflammatoryactivity without associated, systemically-driven, adverse effects. Thecompounds are expected to be useful for a variety of gastrointestinalinflammatory indications that include, but are not limited to,ulcerative colitis (proctosigmoiditis, pancolitis, ulcerative proctitisand left-sided colitis), Crohn's disease, collagenous colitis,lymphocytic colitis, Behcet's disease, celiac disease, checkpoint cancertreatment-induced colitis, (e.g. CTLA-4 inhibitor-induced colitis),ileitis, eosinophilic esophagitis, graft versus host disease-relatedcolitis, and infectious colitis. Ulcerative colitis (Reimund et al., JClin Immunology, 1996, 16, 144-150), Crohn's disease (Woywodt et al.,Eur J Gastroenterology Hepatology, 1999, 11, 267-276), collagenouscolitis (Kumawat et al., Mol Immunology, 2013, 55, 355-364), lymphocyticcolitis (Kumawat et al., 2013), eosinophilic esophagitis(Weinbrand-Goichberg et al., Immunol Res, 2013, 56, 249-260), graftversus host disease-related colitis (Coghill et al., Blood, 2001, 117,3268-3276), infectious colitis (Stallmach et al., Int J Colorectal Dis,2004, 19, 308-315), Behcet's disease (Zhou et al., Autoimmun Rev, 2012,11, 699-704), celiac disease (de Nitto et al., World J Gastroenterol,2009, 15, 4609-4614), checkpoint cancer treatment-induced colitis (e.g.,CTLA-4 inhibitor-induced colitis; (Yano et al., J Translation Med, 2014,12, 191), and ileitis (Yamamoto et al., Dig Liver Dis, 2008, 40,253-259) are characterized by elevation of certain pro-inflammatorycytokine levels. As many pro-inflammatory cytokines signal via JAKactivation, compounds described in this application may be able toalleviate the inflammation and provide symptom relief.

In particular, the compounds of the invention are expected to be usefulfor the induction and maintenance of remission of ulcerative colitis,and for the treatment of Crohn's disease, CTLA-4 inhibitor-inducedcolitis, and the gastrointestinal adverse effects in graft versus hostdisease.

In one aspect, therefore, the invention provides a method of treating agastrointestinal inflammatory disease in a mammal (e.g., a human), themethod comprising administering to the mammal atherapeutically-effective amount of a compound of the invention or of apharmaceutical composition comprising a pharmaceutically-acceptablecarrier and a compound of the invention.

The invention further provides a method of treating ulcerative colitisin a mammal, the method comprising administering to the mammal atherapeutically-effective amount of a compound of the invention or of apharmaceutical composition comprising a pharmaceutically-acceptablecarrier and a compound of the invention.

When used to treat gastrointestinal inflammatory disease, the compoundsof the invention will typically be administered orally in a single dailydose or in multiple doses per day, although other forms ofadministration may be used. The amount of active agent administered perdose or the total amount administered per day will typically bedetermined by a physician, in the light of the relevant circumstances,including the condition to be treated, the chosen route ofadministration, the actual compound administered and its relativeactivity, the age, weight, and response of the individual patient, theseverity of the patient's symptoms, and the like.

Suitable doses for treating ulcerative colitis and othergastrointestinal inflammatory disorders are expected to range from about1 to about 400 mg/day of active agent, including from about 5 to about300 mg/day and from about 20 to about 70 mg per day of active agent foran average 70 kg human.

Combination Therapy

Compounds of the invention may also be used in combination with one ormore agents which act by the same mechanism or by different mechanismsto effect treatment of gastrointestinal inflammatory disorders. Usefulclasses of agents for combination therapy include, but are not limitedto, aminosalicylates, steroids, systemic immunosuppressants, anti-TNFαantibodies, anti-VLA-4 antibodies, anti-integrin α4β7 antibodies,anti-bacterial agents, and anti-diarrheal medicines.

Aminosalicylates that may be used in combination with the present JAKinhibitor compounds include, but are not limited to, mesalamine,osalazine and sulfasalazine. Examples of steroids include, but are notlimited to, prednisone, prednisolone, hydrocortisone, budesonide,beclomethasone, and fluticasone. Systemic immunosuppressants useful fortreatment of inflammatory disorders include, but are not limited tocyclosporine, azathioprine, methotrexate, 6-mercaptopurine, andtacrolimus. Further, anti-TNFα antibodies, which include, but are notlimited to, infliximab, adalimumab, golimumab, and certolizumab, may beused in combination therapy. Useful compounds acting by other mechanismsinclude anti-VLA-4 antibodies, such as natalizumab, anti-integrin α4β7antibodies, such as vedolizumab, anti-bacterial agents, such asrifaximin, and anti-diarrheal medicines, such as loperamide. (Mozaffariet al. Expert Opin. Biol. Ther. 2014, 14, 583-600; Danese, Gut, 2012,61, 918-932; Lam et al., Immunotherapy, 2014, 6, 963-971.)

In another aspect, therefore, the invention provides a therapeuticcombination for use in the treatment of gastrointestinal inflammatorydisorders, the combination comprising a compound of the invention andone or more other therapeutic agents useful for treatinggastrointestinal inflammatory disorders. For example, the inventionprovides a combination comprising a compound of the invention and one ormore agents selected from aminosalicylates, steroids, systemicimmunosuppressants, anti-TNFα antibodies, anti-VLA-4 antibodies,anti-integrin α4β7 antibodies, anti-bacterial agents, and anti-diarrhealmedicines. Secondary agent(s), when included, are present in atherapeutically effective amount, i.e. in any amount that produces atherapeutically beneficial effect when co-administered with a compoundof the invention.

Also provided, therefore, is a pharmaceutical composition comprising acompound of the invention and one or more other therapeutic agentsuseful for treating gastrointestinal inflammatory disorders.

Further, in a method aspect, the invention provides a method of treatinggastrointestinal inflammatory disorders, the method comprisingadministering to the mammal a compound of the invention and one or moreother therapeutic agents useful for treating gastrointestinalinflammatory disorders.

When used in combination therapy, the agents may be formulated in asingle pharmaceutical composition, as disclosed above, or the agents maybe provided in separate compositions that are administeredsimultaneously or at separate times, by the same or by different routesof administration. When administered separately, the agents areadministered sufficiently close in time so as to provide a desiredtherapeutic effect. Such compositions can be packaged separately or maybe packaged together as a kit. The two or more therapeutic agents in thekit may be administered by the same route of administration or bydifferent routes of administration.

Compounds of the invention have been demonstrated to be potentinhibitors of the JAK1, JAK2, JAK3, and TYK2 enzymes in enzyme bindingassays and to have potent functional activity without cytotoxicity incellular assays, as described in the following examples.

EXAMPLES

The following synthetic and biological examples are offered toillustrate the invention, and are not to be construed in any way aslimiting the scope of the invention. In the examples below, thefollowing abbreviations have the following meanings unless otherwiseindicated. Abbreviations not defined below have their generally acceptedmeanings.

-   -   ACN=acetonitrile    -   DCM=dichloromethane    -   DIPEA=N,N-diisopropylethylamine    -   DMF=N,N-dimethylformamide    -   DMSO=dimethyl sulfoxide    -   EtOAc=ethyl acetate    -   h=hour(s)    -   HATU=N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium        hexafluorophosphate    -   min=minute(s)    -   NMP=N-methyl-2-pyrrolidone    -   Pd(dppf)Cl₂=dichloro(1,1′-bis(diphenylphosphino)-ferrocene)dipalladium(II)    -   Pd₂(dba)₃=tris(dibenzylideneacetone)dipalladium(0)    -   PdXPhos=chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II)    -   RT=room temperature    -   Selectfluor=1-chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane        bis(tetrafluoroborate)    -   TEA=triethylamine    -   TFA=trifluoroacetic acid    -   THF=tetrahydrofuran    -   bis(pinacolato)diboron=4,4,5,5,4′,4′,5′,5′-octamethyl-[2,2′]bi[[1,3,2]dioxaborolanyl]    -   Xantphos=4,5-bis(diphenylphosphino)-9,9-dimethylxanthene    -   Xphos=dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl

Reagents and solvents were purchased from commercial suppliers (Aldrich,Fluka, Sigma, etc.), and used without further purification. Progress ofreaction mixtures was monitored by thin layer chromatography (TLC),analytical high performance liquid chromatography (anal. HPLC), and massspectrometry. Reaction mixtures were worked up as described specificallyin each reaction; commonly they were purified by extraction and otherpurification methods such as temperature-, and solvent-dependentcrystallization, and precipitation. In addition, reaction mixtures wereroutinely purified by column chromatography or by preparative HPLC,typically using C18 or BDS column packings and conventional eluents.Typical preparative HPLC conditions are described below.

Characterization of reaction products was routinely carried out by massand ¹H-NMR spectrometry. For NMR analysis, samples were dissolved indeuterated solvent (such as CD₃OD, CDCl₃, or d₆-DMSO), and ¹H-NMRspectra were acquired with a Varian Gemini 2000 instrument (400 MHz)under standard observation conditions. Mass spectrometric identificationof compounds was performed by an electrospray ionization method (ESMS)with an Applied Biosystems (Foster City, Calif.) model API 150 EXinstrument or a Waters (Milford, Mass.)3100 instrument, coupled toautopurification systems.

Preparative HPLC Conditions

Method 1

-   Column: C18, 5 μm 21.2×150 mm or C18, 5 μm 21×250 mm or C14 5 μm    21×150 mm-   Column temperature: Room Temperature-   Flow rate: 20.0 mL/min-   Mobile Phases: A=Water+0.05% TFA    -   B=ACN+0.05% TFA,-   Injection volume: (100-1500 μL)-   Detector wavelength: 214 nm

Crude compounds were dissolved in 1:1 water:acetic acid at about 50mg/mL. A 4 minute analytical scale test run was carried out using a2.1×50 mm C18 column followed by a 15 or 20 minute preparative scale runusing 100 μL injection with the gradient based on the % B retention ofthe analytical scale test run. Exact gradients were sample dependent.Samples with close running impurities were checked with a 21×250 mm C18column and/or a 21×150 mm C14 column for best separation. Fractionscontaining desired product were identified by mass spectrometricanalysis.

Preparative HPLC Conditions

Method 2

-   Column: Synergi 200×50 mm 10 μm-   Column temperature: Room Temperature-   Flow rate: 80 mL/min-   Mobile Phases: A=Water+0.1% TFA    -   B=ACN-   Injection volume: 8 mL-   Detector wavelength: 220 nm and 254 nm-   Gradient: 25% to 45% B    Analytic HPLC Conditions    Method 3-   Column: LUNA C18 (2), 150×4.60 mm, 3 μm-   Column temperature: 37° C.-   Flow rate: 1.0 mL/min-   Injection volume: 5 μL-   Sample preparation: Dissolve in 1:1 ACN:water-   Mobile Phases: A=Water:ACN:TFA (98:2:0.05)    -   B=Water:ACN:TFA (2:98:0.05)-   Detector wavelength: 254 nm-   Gradient: 30 min total (time (min)/% B): 0/2, 10/20, 24/90, 26/90,    27/2, 30/2

Preparation 1:tert-butyl(1R,3s,5S)-3-((7-chloro-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylate

To a 20 mL vial was added 5,7-dichloro-1,6-naphthyridine, (289.1 mg,1.45 mmol), tert-butyl(1R,3s,5S)-3-amino-8-azabicyclo[3.2.1]octane-8-carboxylate (362 mg, 1.60mmol), DIPEA (0.76 mL, 4.36 mmol), and DMSO (7.26 mL). The vial wascapped and the reaction mixture was heated to 110° C. and stirred for 16h. The reaction mixture was diluted with water and extracted with EtOAc(3×20 mL). The combined organic fractions were dried over sodiumsulfate, filtered, and concentrated to afford a brown oil, which waspurified by column chromatography (24 g column; 0-80% EtOAc in hexanes)to afford the title product as a pale yellow solid (455.2 mg, 69% yield;85% purity). (m/z): [M+H]⁺ calcd for C₂₀H₂₅ClN₄O₂ 389.17, 391.16 found391.5.

Preparation 2:N⁵-((1R,3s,5S)-8-azabicyclo[3.2.1]octan-3-yl)-N⁷-(5-methyl-1H-pyrazol-3-yl)-1,6-naphthyridine-5,7-diamine

(a)tert-butyl(1R,3s,5S)-3-((7-((1-(tert-butoxycarbonyl)-5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylate

To a 20 mL vial was added tert-butyl(1R,3s,5S)-3-((7-chloro-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylate(474.6 mg, 1.22 mmol), tert-butyl3-amino-5-methyl-1H-pyrazole-1-carboxylate (289 mg, 1.46 mmol),chloro[2-(dicyclohexylphosphino)-3,6-dimethoxy-2′-4′-6′-tri- (58 mg,0.073 mmol), and cesium carbonate (517 mg, 1.59 mmol). The vial wassealed with a rubber septum and the atmosphere was flushed withnitrogen. Dioxane (6.10 mL) was then added via syringe, and the septumwas quickly replaced with a white cap. The reaction mixture was heatedto 110° C. and stirred for 26 h and cooled to RT. The suspension wasdiluted with water and brine and extracted with EtOAc (4×20 mL). Thecombined organic fractions were dried over sodium sulfate, filtered, andconcentrated to afford a brown foamy solid which was used directly inthe next step. (m/z): [M+H]⁺ calcd for C₂₉H₃₉N₇O₄ 550.31, found 550.8.

(b)N⁵-((1R,3s,5S)-8-azabicyclo[3.2.1]octan-3-yl)-N⁷-(5-methyl-1H-pyrazol-3-yl)-1,6-naphthyridine-5,7-diamine

To the product of the previous step (671 mg, 1.22 mmol) was added DCM(3.05 mL) followed by TFA (3.05 mL) and the reaction mixture was stirredat RT for 4 h and concentrated to afford a thick, red oil. The crude oilwas dissolved in a 15% solution of acetic acid in water including 0.1 mLACN (10 mL) and purified by preparative HPLC (method 1) to afford thedi-TFA salt of the title product as a red/orange solid (705 mg, 73%yield; 97% purity). (m/z): [M+H]⁺ calcd for C₁₉H₂₃N₇ 350.20, found350.5.

Preparation 3:tert-butyl(1R,3s,5S)-3-((7-chloro-1,6-naphthyridin-5-yl)amino)-9-azabicyclo[3.3.1]nonane-9-carboxylate

To a 40 mL vial was added 5,7-dichloro-1,6-naphthyridine, (510 mg, 2.56mmol), tert-butyl(1R,3s,5S)-3-amino-9-azabicyclo[3.3.1]nonane-9-carboxylate (677 mg, 2.82mmol), DIPEA (1.34 mL, 7.69 mmol), and DMSO (8.54 mL). The vial wascapped and the reaction mixture was heated to 110° C. and stirred for 16h. The reaction mixture was diluted with water and brine and extractedwith EtOAc (4×30 mL). The combined organic fractions were dried oversodium sulfate, filtered, and concentrated to afford the desired productas a brown solid which was dissolved in a minimal amount of DCM andadsorbed onto Celite®, purified by column chromatography (40 g column;0-100% EtOAc in hexanes) to afford the title product as a yellow solid(901.6 mg, 86% yield; 99% purity). (m/z): [M+H]⁺ calcd for C₂₁H₂₇ClN₄O₂403.18, found 403.3.

Preparation 4:N⁵-((1R,3s,5S)-9-azabicyclo[3.3.1]nonan-3-yl)-N⁷-(5-methyl-1H-pyrazol-3-yl)-1,6-naphthyridine-5,7-diamine

(a)tert-butyl(1R,3s,5S)-3-((7-((1-(tert-butoxycarbonyl)-5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-9-azabicyclo[3.3.1]nonane-9-carboxylate

To a 100 mL round bottom flask was added the product of Preparation 3(876.4 mg, 2.18 mmol),tert-butyl3-amino-5-methyl-1H-pyrazole-1-carboxylate (515 mg, 2.61mmol), chloro[2-(dicyclohexylphosphino)-3,6-dimethoxy-2′-4′-6′-tri-iso-propyl-1,1′-biphenyl][2-(2-aminoethyl)phenyl]palladium(II)methyl tert-butylether adduct (104 mg, 0.131 mmol), and cesium carbonate(921 mg, 2.83 mmol). The flask was sealed with a rubber septum and theatmosphere was flushed with nitrogen. Dioxane (21.75 mL) was added viasyringe. A condenser with an attached nitrogen balloon was added to theflask, and the reaction mixture was heated to 110° C. and stirred for 20h. The reaction mixture was cooled to RT, diluted with brine, andextracted with EtOAc (4×30 mL). The combined organic fractions weredried over sodium sulfate, filtered, and concentrated to afford a crudebrown solid, which was used in the next step without furtherpurification.

(b)N⁵-((1R,3s,5S)-9-azabicyclo[3.3.1]nonan-3-yl)-N⁷-(5-methyl-1H-pyrazol-3-yl)-1,6-naphthyridine-5,7-diamine

To the product of the previous step (1.226 g, 2.175 mmol) was added DCM(5.44 mL) and TFA (5.44 mL). The flask was covered with a rubber septumpierced with a needle. The solution was stirred at RT for 4 h andconcentrated to afford a dark, red oil. The crude material was dissolvedin a 3:1:0.25 water:acetic acid:acetonitrile solution (18 mL), filtered,and purified in two batches by preparative HPLC to afford the 2TFA saltof the title product as a red solid (991.1 mg, 75% yield; 97% purity).(m/z): [M+H]^(|) calcd for C₂₀H₂₅N₇ 364.22, found 364.1.

Preparation 5: 5,7-dichloro-1,6-naphthyridin-2-ol

(a) N-(2,6-dichloropyridin-4-yl)pivalamide

To a mixture of 2,6-dichloropyridin-4-amine (80.0 g, 491 mmol) and TEA(99.8 g, 982 mmol) in DCM (800 mL) was added pivaloyl chloride (118.0 g,982 mmol) at 0° C., and the mixture was stirred at 20° C. for 13 h,filtered, extracted with DCM, washed with brine, dried over Na₂SO₄,filtered, concentrated in vacuo, and then purified by recrystallizationfrom DCM to afford the title intermediate as a white solid (90 g, 70%yield).

(b) N-(2,6-dichloro-3-formylpyridin-4-yl)pivalamide

To a mixture of the product of the previous step (24.0 g, 97.1 mmol) inTHF (250 mL) was added tert-butyl lithium (224 mL, 291.3 mmol) at −78°C., and the reaction mixture was stirred at −78° C. for 1.5 h. DMF (22.7g, 291.3 mmol) was added, the reaction mixture was stirred at −78° C.for 3 h and 6 M HCl was added. The reaction mixture was extracted withEtOAc, washed with brine, dried, concentrated, and purified by columnchromatography. The products of three identical reactions were combinedto afford the title intermediate (36 g, 45% yield).

(c) tert-butyl3-(2,6-dichloro-4-pivalamidopyridin-3-yl)-3-hydroxypropanoate

Diisopropylamine (17.2 g, 170.8 mmol) was dissolved in THF (100 mL) andcooled to −78° C., and then n-butyl lithium (68.3 mL, 170.8 mmol) wasadded dropwise at −78° C. The mixture was stirred at −78° C. for 0.5 hand then tert-butyl acetate (19.8 g, 170.8 mmol) dissolved in THF (100mL) was added dropwise at −78° C. The reaction mixture was stirred at−78° C. for 0.5 h and then the product of the previous step (18 g, 65.7mmol) dissolved in THF (150 mL) was added dropwise at −78° C. and themixture was stirred at −78° C. for 1.0 h. Aqueous ammonium chloride wasadded and the reaction mixture was extracted with EtOAc (2×800 mL). Theorganic layer was dried and evaporated and the residue was purified bycolumn chromatography. The products of two identical reactions werecombined to provide the title intermediate as a white solid (58 g).

(d) 5,7-dichloro-1,6-naphthyridin-2-ol

Aqueous HCl (400 mL) was added to tert-butyl3-(2,6-dichloro-4-pivalamidopyridin-3-yl)-3-hydroxypropanoate (64 g, 164mmol) dissolved in dioxane (400 mL) and the reaction mixture was stirredat 100° C. for 12 h. Water was added and the reaction mixture wasfiltered to give the title intermediate as a white solid (33 g, 94%yield).

Preparation 6:tert-butyl(1R,3s,5S)-3-((2-(hydroxymethyl)-7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylate

(a)tert-butyl(1R,3s,5S)-3-((7-chloro-2-hydroxy-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylate

To a mixture of 5,7-dichloro-1,6-naphthyridin-2-ol (7.0 g, 32.5 mmol)and tert-butyl(1R,3s,5S)-3-amino-8-azabicyclo[3.2.1]octane-8-carboxylate (8.1 g, 35.8mmol) in DMSO (70 mL) was added DIPEA (8.4 g, 65.0 mmol), The reactionmixture was heated to 110° C. for 8 h, poured into water, filtered, andwashed with EtOAc (200 mL) to give the title intermediate as a yellowsolid (10 g, 75% yield).

(b)tert-butyl(1R,3s,5S)-3-((7-chloro-2-(((trifluoromethyl)sulfonyl)oxy)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylate

To a solution of the product of the previous step (10 g, 24 mmol) andcesium carbonate (15.6 g, 48 mmol) in DMF (100 mL) was added dropwise asolution of N-phenyl-bis(trifluoromethanesulfonimide) (17.0 g, 48 mmol)in DMF (100 mL) at 0° C., and the reaction mixture was stirred at 20° C.for 2 h. Water (200 mL) was added and the reaction mixture was extractedwith EtOAc (400 mL), washed with brine (100 mL), dried over sodiumsulfate, and concentrated to provide the crude title intermediate (14g), which was was used directly in the next step.

(c) Methyl5-(((1R,3s,5S)-8-(tert-butoxycarbonyl)-8-azabicyclo[3.2.1]octan-3-yl)amino)-7-chloro-1,6-naphthyridine-2-carboxylate

To a solution of the product of the previous step (14 g, 26 mmol) inMeOH (280 mL) were added Pd(dppf)Cl₂ (2.0 g, 2.6 mmol) and TEA (5.3 g,52 mmol) and the reaction mixture was heated to 50° C. for 12 h under COatmosphere (50 psi). The reaction mixture was filtered, diluted withwater (100 mL), extracted with EtOAc (300 mL), washed with brine (50mL), dried over sodium sulfate, concentrated and purified by columnchromatography to provide the title intermediate (8.0 g, 70% yield).(m/z): [M+H]⁺ calcd for C₂₂H₂₇ClN₄O₄ 447.17 found 447.1.

(d) Methyl7-((1-(tert-butoxycarbonyl)-5-methyl-1H-pyrazol-3-yl)amino)-5-(((1R,3s,5S)-8-(tert-butoxycarbonyl)-8-azabicyclo[3.2.1]octan-3-yl)amino)-1,6-naphthyridine-2-carboxylate

To a mixture of the product of the previous step (8.0 g, 17.8 mmol),tert-butyl 3-amino-5-methyl-1H-pyrazole-1-carboxylate (4.5 g, 21.5 mmol)and cesium carbonate (7.1 g, 21.5 mmol) in dioxane (80 mL) was addedPdXPhos (2.8 g, 3.56 mmol) under nitrogen. The reaction mixture wasstirred at 100° C. for 12 h, diluted with EtOAc (150 mL), washed withwater (50 mL) and brine (30 mL), dried over anhydrous sodium sulfate,concentrated, and purified by column chromatography to provide the titleintermediate (3.0 g, 72% yield). (m/z): [M+H]⁺ calcd for C₃₁H₄₁N₇O₆608.31 found 608.3.

(e)tert-butyl(1R,3s,5S)-3-((2-(hydroxymethyl)-7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylate

To a solution of sodium borohydride (225 mg, 5.91 mmol) in MeOH (565 mg,17.6 mmol) and THF (10 mL) was added a solution of the product of theprevious step (500 mg, 0.98 mmol) in THF (40 mL) at 0° C., and thereaction mixture was stirred at 50° C. for 2 h. Water (15 mL) was added,followed by EtOAc (150 mL). The reaction mixture was washed with brine(30 mL), dried over anhydrous sodium sulfate, concentrated and purifiedby preparative HPLC (method 2) to afford the title product (840 mg, 46%yield). (m/z): [M+H]⁺ calcd for C₂₅H₃₃N₇O₃ 480.26 found 480.2.

Preparation 7: 2-(Bromomethyl)-5,7-dichloro-1,6-naphthyridine

(a) 2-(2-ethoxy-2-oxoethyl)-6-methylnicotinic acid

To a mixture of potassium tert-butoxide (90.2 g, 804 mmol) in isopropylalcohol (600 mL) was added ethyl acetoacetate (69.8 g, 536 mmol). Thereaction mixture was stirred at RT for 1 h, then copper acetate (4.8 g,26.8 mmol) was added, followed by 2-chloro-6-methylnicotinic acid (46.0g, 268 mmol) and the reaction mixture was stirred at 80° C. for 5 h,dilute HCl was added to adjust the pH to 2 and the solution wasextracted with EtOAc (3×500 mL). The combined organic layers were washedwith brine, dried, and concentrated to give crude product, which wasrecrystallized with 5:1 petroleum ether:toluene to give the titleintermediate as a brown solid (47 g, 79% yield).

(b) 2-Methyl-1,6-naphthyridine-5,7(6H,8H)-dione

To a solution of the product of the previous step (25 g, 105.5 mmol) inTHF (250 mL) was added TEA (15.9 g, 158.2 mmol). The mixture was cooledto −10° C. and ethyl chloroformate (17.2 g, 158.2 mmol) was added, andthe reaction mixture was stirred for 1 h at −10-5° C. Ammonium hydroxide(200 mL) was added dropwise at 0-5° C. and the reaction mixture wasstirred for 2 h at RT. The products of two identical reactions werecombined and the reaction mixture was adjusted to pH 6.5-7.5 with 3 MHCl, concentrated under vacuum, stirred for 1 h at −10-5° C., filtered,and dried under vacuum to give the title intermediate as a brown solid(30 g crude). (m/z): [M+H]⁺ calcd for C₉H₈N₂O₂ 177.07 found 177.1.

(c) 5,7-dichloro-2-methyl-1,6-naphthyridine

The product of the previous step (10.0 g, 56.7 mmol) was dissolved inphosphoryl chloride (40 mL) and stirred for 6 h at 160° C. in a 100 mLsealed tube. The phosphoryl chloride was removed by reduced pressuredistillation and the reaction mixture was poured into ice/water (400 mL)and extracted with DCM (3×400 mL), The organic layers were combined,washed with brine, dried over Na₂SO₄, concentrated, and the residue waspurified by flash chromatography to give the title intermediate productas a red solid (3.4 g, 28% yield).

(d) 2-(Bromomethyl)-5,7-dichloro-1,6-naphthyridine

To a solution of 5,7-dichloro-2-methyl-1,6-naphthyridine (5.0 g, 23.4mmol) in carbon tetrachloride (100 mL) was added N-bromosuccinimide(4.17 g, 23.4 mmol) and benzoyl peroxide (0.28 g, 1.2 mmol) and thereaction mixture was heated at reflux for 12 h and concentrated to givethe crude product which was purified by silica gel chromatography toprovide the title compound as a red solid (3.8 g, 55% yield). (m/z):[M+H]⁺ calcd for C₉H₅BrCl₂N₂ 290.90 found 290.9.

Preparation 8:tert-butyl(1R,3s,5S)-3-((7-chloro-2-(morpholinomethyl)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylate

(a) 4-((5,7-dichloro-1,6-naphthyridin-2-yl)methyl)morpholine

To a solution of 2-(bromomethyl)-5,7-dichloro-1,6-naphthyridine (1.0 g,3.43 mmol) in ACN (34.3 mL) was added DIPEA (1.79 mL, 10.28 mmol)followed by morpholine (0.31 mL, 3.60 mmol) at 0° C. The mixture wasstirred at 0° C. to RT overnight, filtered through a pad of Celite®,which was washed with EtOAc. The combined organic fractions wereconcentrated by rotary evaporation to give crude title product, whichwas used in the next step without purification. (m/z): [M+H]⁺ calcd forC₁₃H₁₃Cl₂N₃O₂ 298.04 found 298.0.

(b)tert-butyl(1R,3s,5S)-3-((7-chloro-2-(morpholinomethyl)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylate

To a mixture of the product of the previous step (1020 mg, 3.42 mmol)and tert-butyl(1R,3s,5S)-3-amino-8-azabicyclo[3.2.1]octane-8-carboxylate (774 mg, 3.42mmol) in DMSO (34.3 mL) was added DIPEA (1.787 mL, 10.26 mmol) at RT.The resulting mixture was stirred at 120° C. for 19 h. Solvent wasremoved by rotary evaporation to give crude product, which was purifiedby column chromatography to give the title compound as a brown solid(513 mg, 30.7% yield). (m/z): [M+H]⁺ calcd for C₂₅H₃₄ClN₅O₃ 488.24 found488.2.

Preparation 9:5,7-dichloro-2-((4,4-difluoropiperidin-1-yl)methyl)-1,6-naphthyridine

Following the general procedure of Preparation 8(a), substituting4,4-difluoropiperidine for morpholine, the title intermediate wasobtained. (m/z): [M+H]⁺ calcd for C₁₄H₁₃Cl₂F₂N₃ 332.05 found 332.0.

Preparation 10:5,7-dichloro-2-((4-methylpiperazin-1-yl)methyl)-1,6-naphthyridine

Following the general procedure of Preparation 8(a), substituting1-methylpiperazine for morpholine, the title intermediate was obtained.(m/z): [M+H]⁺ calcd for C₁₄H₁₆Cl₂N₄ 311.08 found 311.0.

Preparation 11: 5,7-dichloro-3-methoxy-1,6-naphthyridine

(a) 5,7-Dichloro-3-iodo-1,6-naphthyridine

A mixture of 5,7-dichloro-1,6-naphthyridine (4.0 g, 20 mmol) andN-iodosuccinimide (9.0 g, 37 mmol) in acetic acid (100 mL) was heated atreflux for 12 h. The reaction mixture was concentrated under vacuum, andthe residue was purified by column chromatography 20:1 petroleumether:EtOAc to provide the title intermediate as a yellow solid (2.4 g,37% yield).

(b) (5,7-Dichloro-1,6-naphthyridin-3-yl)boronic acid

To a solution of 5,7-dichloro-3-iodo-1,6-naphthyridine (6.0 g, 18.5mmol), bis(pinacolato)diborane (5.2 g, 20.4 mmol) and potassium acetate(3.6 g, 37.0 mmol) in dioxane (50 mL) was added Pd(dppf)₂Cl₂ (0.6 g)under nitrogen. The reaction mixture was heated at 90° C. overnight andfiltered. The filtrate was concentrated to provide the titleintermediate as a yellow oil (6 g, crude).

(c) 5,7-Dichloro-1,6-naphthyridin-3-ol

The product of the previous step (6 g, crude) was dissolved in DCM (20mL), and hydrogen peroxide (6 mL) was added at 0° C. The reactionmixture was stirred at RT overnight and then an aqueous solution ofsodium thiosulfate (20 mL) was added to the mixture at 0° C. The mixturewas extracted with DCM; the combined organic layers were washed withbrine, dried with Na₂SO₄, and concentrated, to provide the titleintermediate as a yellow solid (6.0 g, crude).

(d) 5,7-Dichloro-3-methoxy-1,6-naphthyridine

To a mixture of the product of the previous step (6.0 g, 27.9 mmol) andpotassium carbonate (30.6 g, 83.7 mmol) in DMF (50 mL) was addedmethyliodide (14.4 g, 101 mmol) at RT. The reaction mixture was stirredat RT for 2 h, diluted with water (50 mL), extracted with EtOAc (3×100mL), washed with brine (2×30 mL), dried over Na₂SO₄, filtered andconcentrated under vacuum. The residue was purified by columnchromatography to provide the title compound as a yellow solid (3.0 g,46% yield).

Preparation 12: Methyl5-(((1R,3s,5S)-8-azabicyclo[3.2.1]octan-3-yl)amino)-7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridine-3-carboxylate

(a)tert-Butyl(1R,3s,5S)-3-((7-chloro-3-iodo-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylate

A solution of 5,7-dichloro-3-iodo-1,6-naphthyridine (7.0 g, 21.6 mol),DIPEA (5.6 g, 43.2 mmol) and tert-butyl(1R,3s,5S)-3-amino-8-azabicyclo[3.2.1]octane-8-carboxylate (5.8 g, 25.9mmol) dissolved in NMP (70 mL) was heated at 110° C. for 3 h. Thereaction mixture was purified by column chromatography (eluted withEtOAc:petroleum ether 0-20%) to give the title intermediate as a yellowsolid (10.2 g, 91.8% yield).

(b) Methyl5-(((1R,3s,5S)-8-(tert-butoxycarbonyl)-8-azabicyclo[3.2.1]octan-3-yl)amino)-7-chloro-1,6-naphthyridine-3-carboxylate

The product of the previous step (10.1 g, 19.7 mmol), PdCl₂(dppf) (2.87g, 3.94 mmol) and TEA (5.97 g, 59.2 mmol) was dissolved in methanol (200mL) and the reaction mixture was stirred at 60° C. under CO atmospherefor 4 h, filtered, and evaporated. The residue was purified by columnchromatography (eluted with EtOAc:petroleum ether 0-25%) to give thetitle intermediate as a yellow solid (7.5 g, 85.6% yield).

(c) Methyl5-(((1R,3s,5S)-8-(tert-butoxycarbonyl)-8-azabicyclo[3.2.1]octan-3-yl)amino)-7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridine-3-carboxylate

The product of the previous step (6.6 g, 14.8 mmol), tert-butyl3-amino-5-methyl-1H-pyrazole-1-carboxylate (3.5 g, 17.7 mmol), cesiumcarbonate (9.61 g, 29.6 mmol) and Pd Xphos (2.32 g, 2.95 mmol) weredissolved in dioxane (130 mL) and purged with nitrogen. The reactionmixture was stirred at 110° C. for 12 h. The product was combined withthe product of a similar reaction, extracted with EtOAc (600 mL) andwashed with brine (3×300 mL). The organic layer was evaporated. Theresidue was crystallized to provide the title intermediate (5 g, 58%yield) and 2 g of crude product which was purified by preparative HPLC(method 2).

(d) Methyl5-(((1R,3s,5S)-8-azabicyclo[3.2.1]octan-3-yl)amino)-7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridine-3-carboxylate

The product of the previous step (5.5 g, 10.85 mmol) was dissolved inHCl-methanol (50 mL) and the reaction mixture was stirred at RT for 4 hand evaporated under reduced pressure to give the 2HCl salt of the titlecompound as an orange solid (5.2 g, 100% yield). (m/z): [M+H]⁺ calcd forC₂₁H₂₅N₇O₂ 408.21 found 408.1.

Preparation 13: 5,7-Dichloro-4-methoxy-1,6-naphthyridine

A mixture of 5,7-dichloro-1,6-naphthyridin-4-ol (7 g, 32.6 mmol),methyliodide (41.22 g, 290.4 mmol) and silver carbonate (17.9 g, 65.2mmol) in toluene (140 mL) was stirred at 100° C. for 2 h. The reactionmixture was filtered and concentrated in vacuo and purified by columnchromatography to obtain the title compound as a yellow solid (2.1 g,28.1% yield). (m/z): [M+H]⁺ calcd for C₉H₆Cl₂N₂O 228.99, 230.98 found229.1, 230.0.

Preparation 14: 5,7-dichloro-8-fluoro-1,6-naphthyridine

(a) 2,6-dichloro-3-fluoropyridin-4-amine

To a mixture of 2,6-dichloropyridin-4-amine (3.0 g, 18.4 mmol) in DMF(30 mL) and ACN (30 mL) was added SelectFluor (7.8 g, 22.1 mmol). Thereaction mixture was stirred at 80° C. for 0.5 h, concentrated, andpurified by preparative HPLC (method 2) to give the title intermediateas a white solid (1.5 g, 45% yield). (m/z): [M+H]⁺ calcd for C₅H₃Cl₂FN₂180.97 found 180.9.

(b) N-(2,6-dichloro-3-fluoropyridin-4-yl)pivalamide

To a mixture of the product of the previous step (1.5 g, 8.29 mmol) inTHF (50 mL) was added sodium hydride (662 mg, 16.57 mmol) and pivaloylchloride (1.12 mL, 9.12 mmol) at 0° C. The reaction mixture was stirredat 25° C. for 2 h, diluted with water, extracted with EtOAc (3×100 mL),dried and concentrated to give the title intermediate as a white solid(2.0 g, 90% yield).

(c) 5,7-dichloro-8-fluoro-1,6-naphthyridine

To a solution of the product of the previous step (2.0 g, 7.55) in THF(25 mL) at −70° C. was added a solution of n-butyl lithium (2.5 M, 7.5mL, 18.9 mmol) and the reaction mixture was stirred for 60 min at −10°C. A solution of (E)-3-(dimethylamino)acrylaldehyde (1.12 g, 11.3 mmol)in THF (5 mL) was added over 15 min at −70° C., the reaction mixture wasstirred at −65° C. for 20 min, HCl (5 M, 12 mL) was added and thereaction mixture was stirred at ˜65° C. for 12 h. The reaction mixturewas adjusted to pH 9 with aqueous Na₂CO₃, extracted with EtOAc (3×200mL), dried, and concentrated to give the residue as a brown oil, whichwas purified by column chromatography (eluted with 0-30% EtOAc inpetroleum ether) to give the title compound as a slightly yellow solid(1.0 g, 99% yield). (m/z): [M+H]⁺ calcd for C₈H₃Cl₂FN₂ 216.97, 218.96found 217.0, 219.0.

Preparation 15: 5,7-dichloro-8-fluoro-3-methyl-1,6-naphthyridine

Following the general procedure of Preparation 14 (c) substituting(E)-3-(dimethylamino)-2-methylacrylaldehyde for(E)-3-(dimethylamino)acrylaldehyde, the title intermediate was obtained.(m/z): [M+H]⁺ calcd for C₉H₅Cl₂FN₂ 230.98, 232.98 found 231.0, 233.0.

Preparation 16:tert-Butyl(1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-3-(methylsulfonyl)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylate

(a)tert-Butyl(1R,3s,5S)-3-((7-chloro-3-(methylthio)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylate

A mixture oftert-butyl(1R,3s,5S)-3-((7-chloro-3-iodo-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylate(6.5 g, 12.6 mmol), sodium thiomethoxide (3.0 g, 42.8 mmol), Pd₂(dba)₃(1.1 g, 1.26 mmol), and Xantphos (728 mg, 1.26 mmol) in 3:70water:toluene (73 mL) was stirred at 90° C. for 12 h and concentratedunder vacuum. The residue was purified by column chromatography (1:3EtOAc:petroleum ether) to afford the title intermediate as a yellowsolid (5 g, 90% yield).

(b)tert-Butyl(1R,3s,5S)-3-((7-chloro-3-(methylsulfonyl)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylate

To a solution of the product of the previous step (4.5 g, 10 mmol) inDCM (1000 mL) was added meta-chloroperxoybenzoic acid (5.2 g, 30 mmol)at 0° C. and the reaction mixture was stirred at 0° C. for 3 h, washedwith 10% NaOH (3×200 mL), dried over Na₂SO₄, and filtered. The filtratewas concentrated under vacuum to afford the title intermediate as ayellow solid (4.2 g, 90% yield).

(c)tert-Butyl(1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-3-(methylsulfonyl)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylate

A mixture of the product of the previous step (4.2 g, 9.0 mmol),tert-butyl 3-amino-5-methyl-1H-pyrazole-1-carboxylate (2.7 g, 13.5mmol), PdXphos (1.4 g, 1.8 mmol), and cesium carbonate (5.9 g, 18.0mmol) in dioxane (120 mL) was stirred under nitrogen at 110° C. for 12h, and concentrated under vacuum. The residue was purified by columnchromatography (1:50 methanol:DCM) to afford the title product as ayellow solid (4.0 g, 85% yield). (m/z): [M+H]⁺ calcd for C₂₅H₃₃N₇O₄S528.23 found 528.1.

Preparation 17:tert-Butyl(1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-3-(methylsulfonyl)-1,6-naphthyridin-5-yl)amino)-9-azabicyclo[3.3.1]nonane-9-carboxylate

Following the general procedure of Preparation 16 substitutingtert-butyl(1R,3s,5S)-3-((7-chloro-3-iodo-1,6-naphthyridin-5-yl)amino)-9-azabicyclo[3.3.1]nonane-9-carboxylatefortert-butyl(1R,3s,5S)-3-((7-chloro-3-iodo-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylatein step (a), the title intermediate was prepared as a yellow solid.(m/z): [M+H]⁺ calcd for C₂₆H₃₅N₇O₄S 542.25 found 542.1

Preparation 18:5,7-dichloro-2-((methylsulfonyl)methyl)-1,6-naphthyridine

To a 20 mL vial was added 2-(bromomethyl)-5,7-dichloro-1,6-naphthyridine(307 mg, 1.052 mmol), sodium methanesulfinate 85% (107 mg, 1.052 mmol),and DMF (5.26 mL). The vial was capped and the reaction mixture washeated to 45° C. and stirred for 1.5 h, cooled to RT, and concentratedby rotary evaporation. The solution was diluted with water; a whiteprecipitate formed, which was removed by filtration, and washed withwater and hexanes to afford the title intermediate as a tan solid (259mg, 85% yield). (m/z): [M+H]⁺ calcd for C₁₀H₈Cl₂N₂O₂S 290.97 found290.9.

Example 13-((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)propanenitrile

To a 20 mL vial was addedN⁵-((1R,3s,5S)-8-azabicyclo[3.2.1]octan-3-yl)-N⁷-(5-methyl-1H-pyrazol-3-yl)-1,6-naphthyridine-5,7-diamine,2TFA (462.4 mg, 0.80 mmol), methanol (4 mL), and DIPEA (0.70 mL, 4.00mmol). The reaction mixture was stirred at RT for 5 min, and thenacrylonitrile (0.058 mL, 0.88 mmol, 100 mL) was slowly added. The vialwas capped and the reaction mixture was stirred at RT for 20 h,concentrated, dissolved in a 15% solution of acetic acid in water, andpurified by preparative HPLC (method 1). Fractions were combined,lyophilized, dissolved in 15% acetic acid in water, and purified byreverse-phase HPLC. Fractions were combined and lyophilized to providethe title compound as a bright red solid (505 mg, 52% yield; 98%purity). (m/z): [M+H]⁺ calcd for C₂₂H₂₆N₈ 403.23 found 403.7.

Example 2N⁵-((1R,3s,5S)-8-(2-fluoroethyl)-8-azabicyclo[3.2.1]octan-3-yl)-N⁷-(5-methyl-1H-pyrazol-3-yl)-1,6-naphthyridine-5,7-diamine

To a 250 mL flask was addedN⁵-((1R,3s,5S)-8-azabicyclo[3.2.1]octan-3-yl)-N⁷-(5-methyl-1H-pyrazol-3-yl)-1,6-naphthyridine-5,7-diamine,2 HCl (5.027 g, 11.90 mmol), potassium carbonate (9.87 g, 71.4 mmol),and DMF (59.5 mL). The reaction mixture was stirred at RT for 10 min andthen 1-bromo-2-fluoroethane (1.064 mL, 14.28 mmol) was added in oneportion. The flask was fitted with an air condenser and the reactionmixture was heated to 40° C. and stirred overnight. Another portion of1-bromo-2-fluoroethane was added and the reaction mixture was heated to60° C. and stirred for 24 h. Another portion of 1-bromo-2-fluoroethanewas added, and the reaction mixture was stirred at RT for another 24 h.The suspension was divided into four vials and concentrated by rotaryevaporation to afford the crude product as a red solid. The material ineach vial was dissolved in ˜1:1 water:acetic acid, filtered, andpurified by preparative HPLC (method 1) to afford the desired product(2.31 g total, 48% yield, 97% purity). (m/z): [M+H]⁺ calcd for C₂₁H₂₆FN₇396.22 found 396.2.

Example 3N⁷-(5-methyl-1H-pyrazol-3-yl)-N⁵-((1R,3s,5S)-8-(pyridin-3-ylsulfonyl)-8-azabicyclo[3.2.1]octan-3-yl)-1,6-naphthyridine-5,7-diamine

A mixture of DIPEA (0.815 mL, 4.66 mmol) andN⁵-((1R,3s,5S)-8-azabicyclo[3.2.1]octan-3-yl)-N⁷-(5-methyl-1H-pyrazol-3-yl)-1,6-naphthyridine-5,7-diamine,1TFA (300 mg, 0.78 mmol) in DMF (6 mL) was cooled to 0° C. and thenpyridine-3-sulfonyl chloride (0.093 mL, 0.39 mmol) was added and thesolution was stirred for 20 min. The solvent was removed under vacuumand the crude residue was dissolved in 1:1 acetic acid;water (1 mL) andpurified by reverse phase HPLC to afford the 1TFA salt of the titlecompound as an orange solid (156.5 mg, 33% yield) (m/z): [M+H]⁺ calcdfor C₂₄H₂₆N₈O₂S 491.19 found 491.

Example 42-(Dimethylamino)-1-((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)ethan-1-one

To a 20 mL vial was added dimethylglycine hydrochloride (31 mg, 0.22mmol), HATU (85 mg, 0.22 mmol), and DMF (1 mL). The clear solution wasstirred at RT for 15 min, followed by the addition ofN⁵-((1R,3s,5S)-9-azabicyclo[3.3.1]nonan-3-yl)-N⁷-(5-methyl-1H-pyrazol-3-yl)-1,6-naphthyridine-5,7-diamine2TFA (120 mg, 0.20 mmol). The reaction mixture was stirred at RT for 30min and then DIPEA (0.18 mL, 1.01 mmol) was added. The resultingreaction mixture was stirred at RT for 2 days, and concentrated byrotary evaporation to provide a thick, dark brown oil. The crude oil wasdissolved in 2:1 water:acetic acid, filtered, and purified byreverse-phase HPLC. Fractions were combined and lyophilized to affordthe 2TFA salt of the title compound as a red solid (23 mg, 25% yield.100% purity). (m/z): [M+H]⁺ calcd for C₂₄H₃₂N₈O 449.27 found 449.2.

Example 52,2-difluoro-1-((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)ethan-1-one

To a 20 mL vial was added difluoroacetic acid (0.026 mL, 0.42 mmol),HATU (159 mg, 0.42 mmol), and DMF (1.91 mL). The clear solution wasstirred at RT for 10 min, thenN⁵-((1R,3s,5S)-8-azabicyclo[3.2.1]octan-3-yl)-N⁷-(5-methyl-1H-pyrazol-3-yl)-1,6-naphthyridine-5,7-diamine,2 TFA (220 mg, 0.38 mmol) was added in one portion. The resulting redsolution was stirred at RT for 20 min, and DIPEA (0.33 mL, 1.91 mmol)was added. The vial was capped and the reaction mixture was stirred atRT overnight, and concentrated by rotary evaporation to afford a thick,red oil. The crude oil was dissolved in ˜3:1 water:acetic acid,filtered, and purified by preparative HPLC (method 1) to afford thedesired product as a red solid. Fractions were combined, dissolved in2:1 water:acetic acid and purified by preparative HPLC (method 1) toafford the 1TFA salt of the title product as a red solid (77 mg, 36%yield; 96% purity). (m/z): [M+H]⁺ calcd for C₂₁H₂₃F₂N₇O 428.19 found428.1.

Example 6N⁵-((1R,3s,5S)-8-((2-methoxyethyl)sulfonyl)-8-azabicyclo[3.2.1]octan-3-yl)-N⁷-(5-methyl-1H-pyrazol-3-yl)-1,6-naphthyridine-5,7-diamine

To a 4 mL vial was addedN⁵-((1R,3s,5S)-8-azabicyclo[3.2.1]octan-3-yl)-N⁷-(5-methyl-1H-pyrazol-3-yl)-1,6-naphthyridine-5,7-diamine,2TFA (30 mg, 0.052 mmol), DIPEA (0.045 mL, 0.26 mmol), and DMF (1.15mL). The solution was cooled to 0° C. and a solution of2-methoxy-1-ethanesulfonyl chloride (12 mg, 0.078 mmol) in DMF (1.15 mL)was added slowly to the cold reaction mixture. The vial containing thesulfonyl chloride was rinsed with DMF (1.15 mL) and the rinse was addedto the vial containing the reaction solution, which was capped andstirred at 0° C. for 30 min, warmed to RT, and stirred for 6 h. Thesolution was concentrated by rotary evaporation to afford a red oilwhich was dissolved in 3:1 water: acetic acid, filtered, and purified byreverse-phase HPLC to afford the 1TFA salt of the title compound (12.3mg, 100% purity). (m/z): [M+H]⁺ calcd for C₂₂H₂₉N₇O₃S 472.21 found472.2.

Example 7N⁵-((1R,3s,5S)-8-(ethylsulfonyl)-8-azabicyclo[3.2.1]octan-3-yl)-N⁷-(5-methyl-1H-pyrazol-3-yl)-1,6-naphthyridine-5,7-diamine

Following the procedure of Example 6, using ethylsulfonyl chloride(0.007 mL, 0.078 mmol) in place of 2-methoxy-1-ethanesulfonyl chloride,the 1TFA salt of the title compound (10.1 mg, 100% purity) was prepared.(m/z): [M+H]⁺ calcd for C₂₁H₂₇N₇O₂S 442.20 found 442.2.

Example 8N⁷-(5-methyl-1H-pyrazol-3-yl)-N⁵-((1R,3s,5S)-9-(pyridin-3-ylsulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)-1,6-naphthyridine-5,7-diamine

A mixture ofN⁵-((1R,3s,5S)-9-azabicyclo[3.3.1]nonan-3-yl)-N⁷-(5-methyl-1H-pyrazol-3-yl)-1,6-naphthyridine-5,7-diamine2 HCl (43.6 mg, 0.10 mmol) and DIPEA (0.105 mL, 0.60 mmol) in DMF (1 mL)was cooled to 0° C. and pyridine-3-sulfonyl chloride (17.8 mg, 0.10mmol) was added. The solution was stirred overnight, allowed togradually warm to RT, concentrated to dryness in vacuo, dissolved in 2:1water:acetic acid (1.5 mL), syringe filtered (0.2 micron), and purifiedby reverse phase HPLC to provide the 1TFA salt of the title compound (24mg, 94.4% purity). (m/z): [M+H]⁺ calcd for C₂₅H₂₈N₈O₂S 505.21 found505.2.

Example 9N⁷-(5-methyl-1H-pyrazol-3-yl)-N⁵-((1R,3s,5S)-9-(phenylsulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)-1,6-naphthyridine-5,7-diamine

Following the exact procedure of Example 8, substituting benzensulfonylchloride (17.7 mg, 0.10 mmol) for pyridine-3-sulfonyl chloride (17.8 mg,0.10 mmol), the 1TFA salt of the title compound was obtained (7 mg,94.4% purity). (m/z): [M+H]⁺ calcd for C₂₅H₂₈N₈O₂S 504.21 found 504.1.

Example 10N⁵-((1R,3s,5S)-9-(ethylsulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)-N⁷-(5-methyl-1H-pyrazol-3-yl)-1,6-naphthyridine-5,7-diamine

To a 20 mL vial was addedN⁵-((1R,3s,5S)-9-azabicyclo[3.3.1]nonan-3-yl)-N⁷-(5-methyl-1H-pyrazol-3-yl)-1,6-naphthyridine-5,7-diamine2TFA (100 mg, 0.17 mmol), DIPEA (0.148 mL, 0.85 mmol), and DMF (0.85mL). The solution was cooled to 0° C. and a solution of ethylsulfonylchloride (26 mg, 0.20 mmol) in DMF (0.85 mL) was added to the coldreaction mixture dropwise. The vial containing the sulfonyl chloride wasrinsed with DMF (0.85 mL) and the rinse was added to the vial containingthe reaction solution, which was stirred at 0° C. for 15 min, warmed toRT, and stirred for 20 h. The solution was concentrated by rotaryevaporation to afford a dark red oil which was dissolved in a 3:1water:acetic acid mixture (3 mL), filtered, and purified by preparativeHPLC (method 1) to provide the 1TFA salt of the title compound (13 mg,93% purity). (m/z): [M+H]^(|) calcd for C₂₂H₂₉N₇O₂S 456.21 found 456.1

Example 111-(((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)sulfonyl)azetidine-3-carbonitrile

A mixture ofN⁵-((1R,3s,5S)-9-azabicyclo[3.3.1]nonan-3-yl)-N⁷-(5-methyl-1H-pyrazol-3-yl)-1,6-naphthyridine-5,7-diamine2HCl (58 mg, 0.067 mmol) and DIPEA (0.584 mL, 0.335 mmol) in DMF (1 mL)was cooled to 0° C. and 3-cyano-1-azetidinesulfonyl chloride (24 mg,0.067 mmol) was added. The solution was stirred overnight, allowed togradually warm to RT, concentrated to dryness in vacuo, dissolved in 1:1water:acetic acid (1.5 mL), syringe filtered (0.2 micron), and purifiedby reverse phase HPLC to afford the 1TFA salt of the title compound (7.8mg, 100% purity). (m/z): [M+H]⁺ calcd for C₂₄H₂₉N₉O₂S 508.22 found 508.6

Example 12Isobutyl(1R,3s,5S)-3-((2-(hydroxymethyl)-7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylate

(a)(5-(((1R,3s,5S)-8-azabicyclo[3.2.1]octan-3-yl)amino)-7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-2-yl)methanol

To a 40 mL vial was added tert-butyl(1R,3s,5S)-3-((2-(hydroxymethyl)-7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylate(146.6 mg, 0.31 mmol), 4M HCl in dioxane (1.53 mL, 6.11 mmol), anddioxane (1.53 mL). The vial was capped and the reaction mixture wasstirred at RT for 6 h, frozen at −78° C. and lyophilized to afford the2HCl salt of the title intermediate as a tan solid that was used in nextreaction without purification. (m/z): [M+H]⁺ calcd for C₂₀H₂₅N₇O 380.21found 380.1.

(b)Isobutyl(1R,3s,5S)-3-((2-(hydroxymethyl)-7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylate

To the product of the previous step (138 mg, 0.31 mmol)) was added DMF(1.53 mL), and DIPEA (0.32 mL, 1.83 mmol). The solution was cooled to 0°C. and then isobutyl chloroformate (0.04 mL, 0.31 mmol) was addeddropwise. The vial was capped and the reaction mixture was stirred at 0°C. for 30 min, then warmed to RT, stirred for 1 h, and concentrated byrotary evaporation to afford a red solid. The solid was dissolved in 2:1acetic acid:water, filtered, and purified by preparative HPLC (method 1)to afford the 1TFA salt of the title compound as an orange/red solid(104.2 mg, 57% yield; 99% purity). (m/z): [M+H]⁺ calcd for C₂₅H₃₃N₇O₃480.26 found 480.2.

Example 131-(((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)sulfonyl)azetidine-3-carbonitrile

A mixture ofN⁵-((1R,3s,5S)-8-azabicyclo[3.2.1]octan-3-yl)-N⁷-(5-methyl-1H-pyrazol-3-yl)-1,6-naphthyridine-5,7-diamine2TFA (77 mg, 0.067 mmol) and DIPEA (0.584 mL, 0.335 mmol) in DMF (1 mL)was cooled to 0° C. and 3-cyano-1-azetidinesulfonyl chloride (24 mg,0.067 mmol) was added. The solution was stirred overnight, allowed togradually warm to RT, concentrated to dryness in vacuo, dissolved in 1:1water:acetic acid (1.5 mL), syringe filtered (0.2 micron), and purifiedby reverse phase HPLC to afford the 1TFA salt of the title compound (5.3mg, 100% purity). (m/z): [M+H]⁺ calcd for C₂₃H₂₇N₉O₂S 494.20 found 494.6

Example 14N⁵-((1R,3s,5S)-9-((5-fluoropyridin-3-yl)sulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)-N⁷-(5-methyl-1H-pyrazol-3-yl)-1,6-naphthyridine-5,7-diamine

A mixture ofN⁵-((1R,3s,5S)-9-azabicyclo[3.3.1]nonan-3-yl)-N⁷-(5-methyl-1H-pyrazol-3-yl)-1,6-naphthyridine-5,7-diamine2 HCl (30.2 mg, 0.069 mmol) and DIPEA (0.072 mL, 0.41 mmol) in DMF (1mL) was cooled to 0° C. and 5-fluoropyridine-3-sulfonyl chloride (13.5mg, 0.069 mmol) was added. The solution was stirred overnight, allowedto gradually warm to RT, concentrated to dryness in vacuo, dissolved in2:1 water:acetic acid (1.5 mL), syringe filtered (0.2 micron), andpurified by reverse phase HPLC to provide the 1TFA salt of the titlecompound (20 mg, 100% purity). (m/z): [M+H]⁺ calcd for C₂₅H₂₇FN₈O₂S523.20 found 523.2.

Example 15N⁷⁻(5-methyl-1H-pyrazol-3-yl)-2-(morpholinomethyl)-N⁵-((1R,3s,5S)-8-(2,2,2-trifluoroethyl)-8-azabicyclo[3.2.1]octan-3-yl)-1,6-naphthyridine-5,7-diamine

To a solution ofN⁵-((1R,3s,5S)-8-azabicyclo[3.2.1]octan-3-yl)-N⁷-(5-methyl-1H-pyrazol-3-yl)-2-(morpholinomethyl)-1,6-naphthyridine-5,7-diamine(81 mg, 0.181 mmol) and DIPEA (0.126 mL, 0.722 mmol) in DMF (3.62 mL)wasadded 2,2,2-trifluoroethyltrifluoromethane-sulfonate (0.030 mL, 0.217mmol) at 20° C. The mixture was stirred at 20° C. for 3 days,concentrated by rotary evaporation, and purified by preparative HPLC(method 1) to give the title compound as a red solid (22.1 mg, 19%yield). (m/z): [M+H]⁺ calcd for C₂₆H₃₃F₃N₈O 531.27 found 531.2.

Example 16 Methyl7-((5-methyl-1H-pyrazol-3-yl)amino)-5-(((1R,3s,5S)-8-(2-oxotetrahydro-2H-pyran-3-yl)-8-azabicyclo[3.2.1]octan-3-yl)amino)-1,6-naphthyridine-3-carboxylate

To a solution of methyl5-(((1R,3s,5S)-8-azabicyclo[3.2.1]octan-3-yl)amino)-7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridine-3-carboxylate2HCl in methanol (4 mL) was added tetraalkylammonium carbonate,polymer-bound (0.231 mmol) and the suspension was stirred at RT for 30min, filtered, and concentrated to afford the free-based carboxylatecompound.

To a 20 mL vial was added 3-hydroxytetrahydro-2H-pyran-2-one (0.042 mL,0.46 mmol) and DCM (0.46 mL). The solution was cooled to −40° C., andDIPEA (0.161 mL, 0.92 mmol) was added, followed bytrifluoromethanesulfonic anhydride (0.080 mL, 0.47 mmol). The vial wascapped and stirred at −40° C. for 1 h. After 1 h, a solution of the freebased carboxylate compound (0.094 g, 0.23 mmol) dissolved in DMF (200μL) was added to the cold reaction mixture. The vial was capped and thesolution was stirred at −40° C. and slowly warmed to RT over 2 h,stirred at RT over the weekend, and concentrated by rotary evaporationto afford a thick, brown oil. The crude oil was dissolved in 1:1water:acetic acid, filtered, and purified by reverse-phase HPLC toprovide the title compound as a bright red solid (45 mg, 27% yield; 100%purity). (m/z): [M+H]⁺ calcd for C₂₆H₃₁N₇O₄ 506.24 found 506.1.

Example 17N-methyl-2-((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)acetamide

A solution ofN⁵-((1R,3s,5S)-9-azabicyclo[3.3.1]nonan-3-yl)-N⁷-(5-methyl-1H-pyrazol-3-yl)-1,6-naphthyridine-5,7-diamineTFA (100 mg, 0.209 mmol), 2-bromo-n-methylacetamide (33.4 mg, 0.220mmol), and DIPEA (146 uL) in DMF (2 mL) was stirred overnight,concentrated, and purified by reverse-phase HPLC to provide the titlecompound (50 mg, 55% yield). (m/z): [M+H]⁺ calcd for C₂₃H₃₀N₈O 435.25found 435.6

Using similar synthetic methods, the compounds of Tables 1-8 wereprepared.

TABLE 1

Calc Found Ex No. n R¹ Formula [M + H]⁺ [M + H]⁺ 1-1 1 —CH₂C(O)OCH₃C₂₂H₂₇N₇O₂ 422.22 422.2 1-2 1 —(CH₂)₂C(O)OCH₃ C₂₃H₂₉N₇O₂ 436.24 436.21-3 1 —(CH₂)₃C(O)OCH₃ C₂₄H₃₁N₇O₂ 450.25 450.2 1-4 1 —CH₂C(O)O—iPrC₂₄H₃₁N₇O₂ 450.25 450.2 1-5 1 —CH₂C(O)O—tBu C₂₅H₃₃N₇O₂ 464.27 464.2 1-61 —CH₂C(O)NH₂ C₂₁H₂₆N₈O 407.22 407.2 1-7 1 —CH₂C(O)NHCH₃ C₂₂H₂₈N₈O421.24 421.1 1-8 1 —CH₂C(O)N(CH₃)₂ C₂₃H₃₀N₈O 435.25 435.2 1-9 1—(CH₂)₂CH₂F C₂₂H₂₈FN₇ 410.24 410.2 1-10 1 —(CH₂)₂CF₃ C₂₂H₂₆F₃N₇ 446.22446.2 1-11 1

C₂₅H₃₀N₈O₃ 491.24 491.1 1-12 1

C₂₅H₃₀N₈O₃ 491.24 492.2 1-13 1 —CH₂—iPr C₂₃H₃₁N₇ 406.26 406.2 1-14 1—CH₂CF₃ C₂₁H₂₄F₃N₇ 432.20 432.1 1-15 1 —(CH₂)₃OCH₃ C₂₃H₃₁N₇O 422.26422.1 1-16 1 —CH₂-pyridin-3-yl C₂₅H₂₈N₈ 441.24 441.2 1-17 1—CH₂-pyridin-4-yl C₂₅H₂₈N₈ 441.24 441.2 1-18 1

C₂₆H₂₉N₇O 456.24 456.3 1-19 1

C₂₆H₂₉N₇O 456.24 456.1 1-20 1 —CH₂-tetrahydropyran-4-yl C₂₅H₃₃N₇O 448.27448.2 1-21 1 —CH(CH₃)CH₂CN C₂₃H₂₈N₈ 417.24 417.2 1-22 1 —CH(C₂H₅)CH₂CNC₂₄H₃₀N₈ 431.26 431.2 1-23 1 —CH₂CH(CH₃)CN C₂₃H₂₈N₈ 417.24 417.2 1-24 1

C₂₄H₂₈N₈ 429.24 429.2 1-25 1

C₂₄H₂₈N₈O 445.24 445.1 1-26 1

C₂₃H₂₇N₇O₂ 434.22 434.2 1-27 1

C₂₃H₂₇N₇O₂ 434.22 434.2 1-28 1

C₂₄H₂₉N₇O₂ 448.24 448.2 1-29 1

C₂₄H₂₉N₇O₂ 448.24 448.2 1-30 1

C₂₃H₂₇N₇O₃ 450.22 450.2 1-31 2 —(CH₂)₂CN C₂₃H₂₈N₈ 417.24 417.2 1-32 2—CH₂C(O)OCH₃ C₂₃H₂₉N₇O₂ 436.24 436.2 1-33 2 —(CH₂)₂C(O)OCH₃ C₂₄H₃₁N₇O₂450.25 450.2 1-34 2 —(CH₂)₃C(O)OCH₃ C₂₅H₃₃N₇O₂ 464.27 464.2 1-35 2—CH₂C(O)O—iPr C₂₅H₃₃N₇O₂ 464.27 464.2 1-36 2 —CH₂C(O)O—tBu C₂₆H₃₅N₇O₂478.29 478.2 1-37 2 —CH₂C(O)NH₂ C₂₂H₂₈N₈O 421.24 421.2 1-38 2—CH₂C(O)N(CH₃)₂ C₂₄H₃₂N₈O 449.27 449.2 1-39 2 —CH₂CH₂F C₂₂H₂₈FN₇ 410.24410.2 1-40 2 —(CH₂)₂CH₂F C₂₃H₃₀FN₇ 424.25 424.2 1-41 2

C₂₆H₃₂N₈O₃ 505.26 505.2 1-42 2

C₂₆H₃₂N₈O₃ 505.26 505.2 1-43 2 —CH₂—iPr C₂₄H₃₃N₇ 420.28 420.2 1-44 2—(CH₂)₂CF₃ C₂₃H₂₈F₃N₇ 460.24 460.2 1-45 2 —CH₂CF₃ C₂₂H₂₆F₃N₇ 446.22446.1 1-46 2 —(CH₂)₃OCH₃ C₂₄H₃₃N₇O 436.27 436.2 1-47 2 —CH₂-pyridin-3-ylC₂₆H₃₀N₈ 455.26 455.2 1-48 2 —CH₂-pyridin-4-yl C₂₆H₃₀N₈ 455.26 455.21-49 2

C₂₇H₃₁N₇O 470.26 470.1 1-50 2

C₂₇H₃₁N₇O 470.26 470.3 1-51 2

C₂₄H₂₉N₇O₂ 448.24 448.2 1-52 2

C₂₄H₂₉N₇O₂ 448.24 448.2 1-53 2

C₂₅H₃₁N₇O₂ 462.25 462.2 1-54 2

C₂₇H₂₉N₉ 480.25 480.2 1-55 2

C₂₇H₂₉N₉ 480.25 480.2 1-56 2

C₂₇H₂₉N₉ 480.25 480.2 1-57 2

C₂₅H₃₁N₇O₂ 462.25 462.2 1-58 2

C₂₄H₂₉N₇O₃ 464.23 464.2 (a) Stereoisomers separated but not identified

TABLE 2

Calc Found Ex No. n R⁶ Formula [M + H]⁺ [M + H]⁺ 2-1 1 —C₂H₅ C₂₂H₂₇N₇O406.23 406.2 2-2 1 —CH₂F C₂₁H₂₄FN₇O 410.20 410.1 2-3 1 —CH(CH₃)FC₂₂H₂₆FN₇O 424.22 424.1 2-4 1 —CH₂OC₂H₅ C₂₃H₂₉N₇O₂ 436.24 436.2 2-5 1—CH₂N(CH₃)₂ C₂₃H₃₀N₈O 435.25 435.2 2-6 1 —CH((S)CH₃)C₂H₅ C₂₄H₃₁N₇O434.26 434.2 2-7 1

C₂₄H₂₆F₃N₇O 486.22 486.2 2-8 1

C₂₄H₂₆N₈O 443.22 443.2 2-9 1

C₂₅H₂₉N₇O₃ 476.23 476.2 2-10 1

C₂₆H₃₃N₇O 460.27 460.6 2-11 1 —CH₂-phenyl C₂₇H₂₉N₇O 468.24 468.1 2-12 1phenyl C₂₆H₂₇N₇O 454.23 454.2 2-13 1 pyridin-4-yl C₂₅H₂₆N₈O 455.22 455.12-14 1 pyridin-3-yl C₂₅H₂₆N₈O 455.22 455.5 2-15 2 —C₂H₅ C₂₃H₂₉N₇O 420.24420.2 2-16 2 —CH((S)OH)CH₃ C₂₃H₂₉N₇O₂ 436.24 436.2 2-17 2 —CH₂OC₂H₅C₂₄H₃₁N₇O₂ 450.25 450.2 2-18 2 —CH((S)CH₃)C₂H₅ C₂₅H₃₃N₇O 448.27 448.22-19 2

C₂₅H₂₈F₃N₇O 500.23 500.2 2-20 2

C₂₅H₂₈N₈O 457.24 457.2 2-21 2

C₂₆H₃₁N₇O₃ 490.25 490.2 2-22 2 —C(CH₃)₂CH₂OH C₂₅H₃₃N₇O₂ 464.27 464.22-23 2

C₂₇H₃₅N₇O 474.29 474.6 2-24 2 —CH₂-phenyl C₂₈H₃₁N₇O 482.26 482.1 2-25 2phenyl C₂₇H₂₉N₇O 468.24 468.1 2-26 2

C₂₇H₂₇N₉O 494.23 494.2 2-27 2 —(CH₂)₃CN C₂₅H₃₀N₈O 459.25 459.2 2-28 2

C₂₇H₂₇N₉O 494.23 494.2 2-29 2 —CH(CH₃)CH₂CN C₂₅H₃₀N₈O 459.25 459.2 2-302

C₂₇H₂₇N₉O 494.23 494.2 2-31 2 —(CH₂)₂—iPr C₂₆H₃₅N₇O 462.29 462.2

TABLE 3

Calc Found Ex No. n R⁸ Formula [M + H]⁺ [M + H]⁺ 3-1 1 —C₂H₅ C₂₂H₂₇N₇O₂422.22 422.2 3-2 1 —iPr C₂₃H₂₉N₇O₂ 436.24 436.2 3-3 1 —CH₂—iPrC₂₄H₃₁N₇O₂ 450.25 450.2 3-4 1 —CH₂CH═CH₂ C₂₃H₂₇N₇O₂ 434.22 434.2 3-5 1—(CH₂)₂OCH₃ C₂₃H₂₉N₇O₃ 452.23 452.1 3-6 2 —C₂H₅ C₂₃H₂₉N₇O₂ 436.24 436.23-7 2 —CH₂—iPr C₂₅H₃₃N₇O₂ 464.27 464.2 3-8 2 —CH₂CH═CH₂ C₂₄H₂₉N₇O₂448.24 448.2 3-9 2 —(CH₂)₂OCH₃ C₂₄H₃₁N₇O₃ 466.25 466.2 3-10 2—CH₂-cpropyl C₂₅H₃₁N₇O₂ 462.25 462.2 3-11 2 —CH₂CN C₂₄H₂₇N₇O₂ 446.22446.2 3-12 2 —CH(CH₃)C₂H₅ C₂₅H₃₃N₇O₂ 464.27 464.2 3-13 2 —CH₂-C₂₆H₃₃N₇O₃ 492.26 492.2 tetrahydrofuran- 2-yl 3-14 2 —(CH₂)₂CNC₂₅H₂₉N₇O₂ 460.24 460.2

TABLE 4

Calc Found Ex No. n R⁹ Formula [M + H]⁺ [M + H]⁺ 4-1 1 —CH₃ C₂₀H₂₅N₇O₂S428.18 428.2 4-2 1 —iPr C₂₂H₂₉N₇O₂S 456.21 456.2 4-3 1 —CH₂—iPrC₂₃H₃₁N₇O₂S 470.23 470.2 4-4 1 cyclopropyl C₂₂H₂₇N₇O₂S 454.20 454.2 4-51 cyclopentyl C₂₄H₃₁N₇O₂S 482.23 482.2 4-6 1 azetidin-1-yl C₂₂H₂₈N₈O₂S469.21 469.2 4-7 1

C₂₄H₃₁N₇O₂S 482.23 482.2 4-8 1

C₂₃H₂₉N₇O₂S 468.21 468.2 4-9 1 azetidin-3-yl C₂₂H₂₈N₈O₂S 469.21 469.24-10 1

C₂₄H₃₂N₈O₂S 497.24 497.2 4-11 1

C₂₅H₃₁N₉O₂S 522.23 522.2 4-12 1

C₂₅H₃₄N₈O₃S 527.25 527.6 4-13 1 —CH₂-phenyl C₂₆H₂₉N₇O₂S 504.21 504.14-14 1 pyridin-2-yl C₂₄H₂₆N₈O₂S 491.19 491.1 4-15 1 5-fluoropyridin-3-ylC₂₄H₂₅FN₈O₂S 509.18 509.1 4-16 2 —CH₃ C₂₁H₂₇N₇O₂S 442.20 442.2 4-17 2—CH₂—iPr C₂₄H₃₃N₇O₂S 484.24 484.2 4-18 2 cyclopropyl C₂₃H₂₉N₇O₂S 468.21468.2 4-19 2 —(CH₂)₂OCH₃ C₂₃H₃₁N₇O₃S 486.22 486.2 4-20 2

C₂₅H₃₃N₇O₂S 496.24 496.2 4-21 2 azetidin-3-yl C₂₃H₃₀N₈O₂S 483.22 483.24-22 2 —CH₂CH═CH₂ C₂₃H₂₉N₇O₂S 468.21 468.6 4-23 2 —CH₂-cyclopropylC₂₄H₃₁N₇O₂S 482.23 482.6 4-24 2 —CH₂-phenyl C₂₇H₃₁N₇O₂S 518.23 518.14-25 2 4-methylpyridin-3-yl C₂₆H₃₀N₈O₂S 519.22 519.2 4-26 2—CH₂-pyridin-3-yl C₂₆H₃₀N₈O₂S 519.22 519.2 4-27 2

C₂₄H₃₂N₈O₂S 497.24 497.6 4-28 2

C₂₆H₃₆N₈O₃S 541.26 541.8 4-29 2 azetidin-1-yl C₂₃H₃₀N₈O₂S 483.22 483.2

TABLE 5

Calc Found Ex No. n R² R¹ Formula [M + H]⁺ [M + H]⁺ 5-1 1 —OCH₃ —CH₂CF₃C₂₂H₂₆F₃N₇O 462.22 462.1 5-2 1 —OCH₃ —(CH₂)₃CN C₂₄H₃₀N₈O 447.25 447.25-3 1 —OCH₃ —CH₂CH₂F C₂₂H₂₈FN₇O 426.23 426.2 5-4 1 —OCH₃ —CH₂—iPrC₂₄H₃₃N₇O 436.27 436.2 5-5 1 —OCH₃ —(CH₂)₂CH₂F C₂₃H₃₀FN₇O 440.25 440.15-6 1 —OCH₃

C₂₅H₂₈F₃N₇O₂ 516.23 516 5-7 1 —OCH₃ —C(O)OCH₂CH═CH₂ C₂₄H₂₉N₇O₃ 464.23464 5-8 1 —OCH₃ —(CH₂)₂CN C₂₃H₂₈N₈O 433.24 433.1 5-9 1 —OCH₃ —S(O)₂CH₃C₂₁H₂₇N₇O₃S 458.19 458 5-10 2 —OCH₃ —(CH₂)₂CN C₂₄H₃₀N₈O 447.25 447.15-11 1 —CH₂OH —(CH₂)₂CN C₂₃H₂₈N₈O 433.24 433.2 5-12 1 —CH₂OH —S(O)₂CH₃C₂₁H₂₇N₇O₃S 458.19 458.1 5-13 1 —CH₂OH —S(O)₂C₂H₅ C₂₂H₂₉N₇O₃S 472.21472.1 5-14 1 —CH₂OH —(CH₂)₂CH₂F C₂₂H₂₈FN₇O 426.23 426.2 5-15 1

—S(O)₂CH₃ C₂₅H₃₄N₈O₃S 527.25 527.6 5-16 1

—S(O)₂C₂H₅ C₂₆H₃₆N₈O₃S 541.26 541.8 5-17 1

—C(O)OCH₂CH═CH₂ C₂₈H₃₆N₈O₃ 533.29 533.8 5-18 1

—(CH₂)₂CN C₂₇H₃₅N₉O 502.30 502.8 5-19 1

—CH₂—iPr C₂₈H₄₀N₈O 505.33 505.3 5-20 1

C₂₉H₃₄FN₉O₃S 608.25 608 5-21 1

C₂₇H₃₇N₉O₃S 568.27 568.2 5-22 2

—S(O)₂CH₃ C₂₆H₃₆N₈O₃S 541.26 541.8 5-23 2

—S(O)₂C₂H₅ C₂₇H₃₈N₈O₃S 555.28 555.8 5-24 2

—S(O)₂(CH₂)₂OCH₃ C₂₈H₄₀N₈O₄S 585.29 585.8 5-25 2

—C(O)OCHCH═CH₂ C₂₉H₃₈N₈O₃ 547.31 547.8 5-26 2

—(CH₂)₂CN C₂₈H₃₇N₉O 516.31 516.8 5-27 2

—S(O)₂-pyridin-3-yl C₃₀H₃₇N₉O₃S 604.27 604.1 5-28 2

—S(O)₂-azetidin-1-yl C₂₈H₃₉N₉O₃S 582.29 582.1 5-29 2

—CH₂CF₃ C₂₇H₃₅F₃N₈O 545.29 545.2 5-30 2

C₃₀H₃₆FN₉O₃S 622.26 622.2 5-31 1

C₃₀H₃₄F₃N₉O₂S 642.25 642 5-32 1

C₃₀H₄₀F₂N₈O₂S 615.30 615 5-33 1

—CH₂CF₃ C₂₇H₃₃F₅N₈ 565.28 565.2 5-34 1

—CH₂—iPr C₂₉H₄₀F₂N₈ 539.33 539.3 5-35 1

—CH₂CF₃ C₂₇H₃₆F₃N₉ 544.30 544.3 5-36 1

C₃₀H₄₃N₉O₂S 594.33 594.3 5-37 1

—(CH₂)₂CN C₂₈H₃₈N₁₀ 515.33 515.3 5-38 1

—CH₂—iPr C₂₉H₄₃N₉ 518.36 518.3 5-39 1

C₂₈H₄₀N₁₀O₂S 581.31 581.3 5-40 1

C₃₀H₃₇FN₁₀O₂S 621.28 621.2 5-41 1 —CH₂S(O)₂CH₃ —CH₂CH₂F C₂₃H₃₀FN₇O₂S488.22 488.1 5-42 1 —CH₂S(O)₂CH₃ —(CH₂)₂CH₂F C₂₄H₃₂FN₇O₂S 502.23 502.15-43 1 —CH₂S(O)₂CH₃ —(CH₂)₂CF₃ C₂₄H₃₀F₃N₇O₂S 538.21 538.1

TABLE 6

Calc Found Ex No. n R³ R¹ Formula [M + H]⁺ [M + H]⁺ 6-1 1 —CH₃ —(CH₂)₂CNC₂₃H₂₈N₈ 417.24 417.2 6-2 1 —CH₃ —CH₂-pyridin-4-yl C₂₆H₃₀N₈ 455.26 455.26-3 1 —CH₃ —C(O)OCH₂—iPr C₂₅H₃₃N₇O₂ 464.27 464.2 6-4 1 —CH₃

C₂₄H₂₉N₉O₂S 508.22 508.2 6-5 1 —CH₃ —S(O)₂CH₃ C₂₁H₂₇N₇O₂S 442.20 442.16-6 2 —CH₃ —(CH₂)₂CN C₂₄H₃₀N₈ 431.26 431.2 6-7 2 —CH₃ —C(O)OCH₂—iPrC₂₆H₃₅N₇O₂ 478.29 478.2 6-8 2 —CH₃ —S(O)₂CH₃ C₂₂H₂₉N₇O₂S 456.21 456.26-9 2 —CH₃

C₂₅H₃₁N₉O₂S 522.23 522.1 6-10 2 —CH₃ —CH₂-pyridin-4-yl C₂₇H₃₂N₈ 469.28469.3 6-11 1 —OCH₃ —(CH₂)₂CN C₂₃H₂₈N₈O 433.24 433.1 6-12 1 —OCH₃—(CH₂)₃CN C₂₄H₃₀N₈O 447.25 447.2 6-13 1 —OCH₃ —CH₂CH₂F C₂₂H₂₈FN₇O 426.23426.1 6-14 1 —OCH₃ —CH₂—iPr C₂₄H₃₃N₇O 436.27 436.2 6-15 1 —OCH₃—(CH₂)₂CF₃ C₂₃H₂₈F₃N₇O 476.23 476.1 6-16 1 —OCH₃ —(CH₂)₂CH₂F C₂₃H₃₀FN₇O440.25 440.2 6-17 1 —OCH₃ —CH₂CF₃ C₂₂H₂₆F₃N₇O 462.22 462.1 6-18 1 —OCH₃—S(O)₂CH₃ C₂₁H₂₇N₇O₃S 458.19 458.1 6-19 1 —OCH₃ —C(O)OCH₂CH═CH₂C₂₄H₂₉N₇O₃ 464.23 464.1 6-20 1 —OCH₃

C₂₄H₂₉N₉O₃S 524.21 524.1 6-21 1 —OCH₃ —S(O)₂(CH₂)₃CN C₂₄H₃₀N₈O₃S 511.22511.1 6-22 1 —OCH₃ —S(O)₂(CH₂)₂CN C₂₃H₂₈N₈O₃S 497.20 497.2 6-23 1 —OCH₃

C₂₅H₃₀N₈O₃S 523.22 523.2 6-24 1 —OCH₃ —CH₂-pyridin-4-yl C₂₆H₃₀N₈O 471.25471.2 6-25 1 —OCH₃ —C(O)OCH₂—iPr C₂₅H₃₃N₇O₃ 480.26 480.2 6-26 2 —OCH₃—(CH₂)₂CH₂F C₂₄H₃₂FN₇O 454.27 454.2 6-27 2 —OCH₃ —C(O)CH((S)OH)CH₃C₂₄H₃₁N₇O₃ 466.25 466.2 6-28 2 —OCH₃ —CH₂-pyridin-4-yl C₂₇H₃₂N₈O 485.27485.2 6-29 2 —OCH₃ —S(O)₂CH₃ C₂₂H₂₀N₇O₃S 472.21 472.1 6-30 2 —OCH₃—C(O)CH₂N(CH₃)₂ C₂₅H₃₄N₈O₂ 479.28 479.3 6-31 2 —OCH₃ —(CH₂)₂CN C₂₄H₃₀N₈O447.25 447.2 6-32 1 —C(O)OCH₃ —(CH₂)₂CN C₂₄H₂₈N₈O₂ 461.23 461.2 6-33 1—C(O)OCH₃ —S(O)₂CH₃ C₂₂H₂₇N₇O₄S 486.18 468.1 6-34 1 —C(O)OCH₃ —S(O)₂C₂H₅C₂₃H₂₀N₇O₄S 500.20 500.2 6-35 1 —C(O)OCH₃

C₂₅H₂₀N₇O₄ 492.23 492.1 6-36 1 —C(O)OCH₃ —C(O)C₂H₅ C₂₄H₂₉N₇O₃ 464.23464.2 6-37 1 —C(O)OCH₃ —C(O)CHF₂ C₂₃H₂₅F₂N₇O₃ 486.20 468.2 6-38 1—C(O)OCH₃ —C(O)CH((S)CH₃)C₂H₅ C₂₆H₃₃N₇O₃ 492.26 492.2 6-39 1 —C(O)OCH₃—C(O)CH₂N(CH₃)₂ C₂₅H₃₂N₈O₃ 493.26 493.2 6-40 1 —C(O)OCH₃ —C(O)OCH₂CH═CH₂C₂₅H₂₉N₇O₄ 492.23 492.2 6-41 1 —C(O)OCH₃ —CH₂C(O)O—iPr C₂₆H₃₃N₇O₄ 508.26508.2 6-42 1 —C(O)OCH₃ —CH₂C(O)NHCH₃ C₂₄H₃₀N₈O₃ 479.24 479.5 6-43 1—C(O)OCH₃ —CH₂CH₂F C₂₃H₂₈FN₇O₂ 454.23 454.2 6-44 1 —C(O)OCH₃ —(CH₂)₂CH₂FC₂₄H₃₀FN₇O₂ 468.24 468.2 6-45 1 —C(O)OCH₃ —(CH₂)₂CF₃ C₂₄H₂₈F₃N₇O₂ 504.23504.2 6-46 1 —C(O)OCH₃

C₂₆H₃₁N₇O₄ 506.24 507.2 6-47 2 —C(O)OCH₃ —(CH₂)₂CN C₂₅H₃₀N₈O₂ 475.25475.2 6-48 2 —C(O)OCH₃

C₂₇H₃₃N₇O₄ 520.26 520.2 6-49 2 —C(O)OCH₃

C₂₇H₃₃N₇O₄ 520.26 520.2 6-50 1 —S(O)₂CH₃ —(CH₂)₂CN C₂₃H₂₈N₈O₂S 481.21481.2 6-51 1 —S(O)₂CH₃ —C(O)CH₂CN C₂₃H₂₆N₈O₃S 495.19 495.2 6-52 1—S(O)₂CH₃ —C(O)CHF₂ C₂₂H₂₅F₂N₇O₃S 506.17 506.2 6-53 1 —S(O)₂CH₃—CH((S)CH₃)C₂H₅ C₂₅H₃₃N₇O₃S 512.24 512.2 6-54 1 —S(O)₂CH₃ —CH₂CH₂FC₂₂H₂₈FN₇O₂S 474.20 474.2 6-55 1 —S(O)₂CH₃ —(CH₂)₂CH₂F C₂₃H₃₀FN₇O₂S488.22 488.2 6-56 1 —S(O)₂CH₃ —(CH₂)₂CF₃ C₂₃H₂₈F₃N₇O₂S 524.20 524.2 6-571 —S(O)₂CH₃

C₂₄H₂₉N₇O₄S 512.20 512.1 6-58 1 —S(O)₂CH₃

C₂₄H₂₉N₇O₄S 512.20 512.2 6-59 1 —S(O)₂CH₃ —CH₂-pyridin-4-yl C₂₆H₃₀N₈O₂S519.22 519.1 6-60 2 —S(O)₂CH₃ —CH₂-pyridin-4-yl C₂₇H₃₂N₈O₂S 533.24 533.1(a) Stereoisomers separated but not identified

TABLE 7

Calc Found Ex No. n R⁴ R¹ Formula [M + H]⁺ [M + H]⁺ 7-1 1 —OCH₃—C(O)OCH₂—iPr C₂₅H₃₃N₇O₃ 480.26 480.1 7-2 1 —OCH₃ —S(O)₂C₂H₅ C₂₂H₂₀N₇O₃S472.21 472.1 7-3 1 —OCH₃ —C(O)CH₂N(CH₃)₂ C₂₄H₃₂N₈O₂ 465.27 465.1 7-4 1—OCH₃ —C(O)CH(CH₃)C₂H₅ C₂₅H₃₃N₇O₂ 464.27 464.2 7-5 1 —OCH₃ —C(O)CHF₂C₂₂H₂₅F₂N₇O₂ 458.20 458.1 7-6 1 —OCH₃ —(CH₂)₂CN C₂₃H₂₈N₈O 433.24 433.27-7 1 —OCH₃ —CH₂CH₂F C₂₂H₂₈FN₇O 426.23 426.2 7-8 1 —OCH₃ —CH₂CHF₂C₂₂H₂₇F₂N₇O 444.22 444.1 7-9 1 —OCH₃ —CH₂CF₃ C₂₂H₂₆F₃N₇O 462.22 462.17-10 1 —OCH₃

C₂₅H₃₀N₈O₂ 475.25 475.2

TABLE 8

Calc Found Ex No. n R³ R¹ Formula [M + H]⁺ [M + H]⁺ 8-1 1 H —(CH₂)₂CNC₂₂H₂₅FN₈ 421.22 421.2 8-2 1 H —S(O)₂CH₃ C₂₀H₂₄FN₇O₂S 446.17 446.1 8-3 1H —S(O)₂C₂H₅ C₂₁H₂₆FN₇O₂S 460.19 460.2 8-4 1 H —S(O)₂-pyridin-3-ylC₂₄H₂₅FN₈O₂S 509.18 509.2 8-5 1 H

C₂₄H₂₄F₂N₈O₂S 527.17 527.1 8-6 1 H —CH₂-pyridin-4-yl C₂₅H₂₇FN₈ 459.23459.2 8-7 1 H —C(O)CH₂N(CH₃)₂ C₂₃H₂₉FN₈O 453.25 453.2 8-8 1 H—C(O)CH((S)OH)CH₃ C₂₂H₂₆FN₇O₂ 440.21 440.2 8-9 2 H —(CH₂)₂CN C₂₃H₂₇FN₈435.23 435.2 8-10 2 H —S(O)₂CH₃ C₂₁H₂₆FN₇O₂S 460.19 460.6 8-11 2 H—S(O)₂-phenyl C₂₆H₂₈FN₇O₂S 522.20 522.2 8-12 2 H —S(O)₂-pyridin-3-ylC₂₅H₂₇FN₈O₂S 523.20 523.1 8-13 2 H

C₂₅H₂₆F₂N₈O₂S 541.19 541.1 8-14 2 H —CH₂-pyridin-4-yl C₂₆H₂₉FN₈ 473.25473.2 8-15 2 H —C(O)CH₂N(CH₃)₂ C₂₄H₃₁FN₈O 467.26 467.2 8-16 2 H—C(O)CH((S)OH)CH₃ C₂₃H₂₈FN₇O₂ 454.23 454.2 8-17 1 —CH₃ —S(O)₂CH₃C₂₁H₂₆FN₇O₂S 460.19 460.1 8-18 1 —CH₃ —(CH₂)₂CN C₂₃H₂₇FN₈ 435.23 435.18-19 1 —CH₃

C₂₄H₂₈FN₉O₂S 526.21 526.1 8-20 1 —CH₃ —S(O)₂-pyridin-3-yl C₂₅H₂₇FN₈O₂S523.20 523.1 8-21 2 —CH₃ —(CH₂)₂CN C₂₄H₂₉FN₈ 449.25 449.2 8-22 2 —CH₃—S(O)₂CH₃ C₂₂H₂₈FN₇O₂S 474.20 474.1 8-23 2 —CH₃

C₂₅H₃₀FN₉O₂S 540.22 540.2 8-24 2 —CH₃ —S(O)₂-pyridin-3-yl C₂₆H₂₉FN₈O₂S537.21 537.1

Example 18 Crystalline solvate3-((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)propanenitrileForm II (a)tert-butyl((1R,3s,5S)-3-((7-chloro-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylate

To a 2 L flask was added 5,7-dichloro-1,6-naphthyridine (45.8 g, 230mmol), tert-butyl(1R,3s,5S)-3-amino-8-azabicyclo[3.2.1]octane-8-carboxylate (54.7 g, 242mmol), and DMSO (458 mL) followed by DIPEA (64.3 mL, 368 mmol). Thereaction mixture was heated at 110° C. for 12 h, cooled to ambienttemperature and water (458 mL) was added slowly over 45 min. After 2 h,the reaction mixture was filtered and washed with 1:1 DMSO:water (60 mL)to give a wet solid product (65 g). The solid was washed in fourportions with MTBE (500 mL) to give the title intermediate (74 g, 190mmol, 83% yield) (HPLC Method 3 Retention time 20.20 min) and thefiltrate which was concentrated to give a solid (19.5 g), mainlyproduct. Heptane (195 mL) was added to the solid (19.5 g) and thereaction mixture was stirred for 2 h, filtered and washed with heptaneto give another portion of the title intermediate (12.3 g, 31.6 mmol,13.75% yield) HPLC Method 3 Retention time 20.20 min.

(b)tert-butyl((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylate

A mixture of tert-butyl((1R,3s,5S)-3-((7-chloro-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylate(30 g, 77 mmol), tert-butyl 3-amino-5-methyl-1H-pyrazole-1-carboxylate(19.78 g, 100 mmol), Cs₂CO₃ (50.3 g, 154 mmol), PdXPhos (1.517 g, 1.93mmol) and XPhos (0.919 g, 1.93 mmol) was degassed with nitrogen 3 times,and then 1,4-dioxane (300 mL) was added. The reaction mixture wasdegassed with nitrogen 5 times, heated to reflux, stirred for 16 h, andcooled to 75° C. Water (90 mL) was added and the reaction mixture washeated to reflux and stirred at reflux for 48 h. Water (210 mL) wasadded slowly and the reaction mixture was stirred at RT fort 1 h, andfiltered. The filter cake was washed with 1:1 dioxane:water (50 mL) anddried at 50° C. under vacuum overnight to give crude title intermediate(35.34 g).

The crude product was dissolved in DMF (173 mL). SiliaMetS® thiolfunctionalized silica (8.65 g) was added and the reaction mixture wasstirred for 45 min at 80° C., cooled to RT, filtered, and rinsed withDMF (35 mL). To the filtrate was added water (346 mL) dropwise and seedsfrom a previous preparation by the same procedure. The reaction mixturestirred at RT for 6 h, filtered, washed with water (35 mL) and dried at50° C. under vacuum to give the title intermediate (33.6 g, 97% yield).HPLC Method 3 Retention time 16.89 min

(c)N⁵-((1R,3s,5S)-8-azabicyclo[3.2.1]octan-3-yl)-N⁷-(5-methyl-1H-pyrazol-3-yl)-1,6-naphthyridine-5,7-diamine

To a suspension of tert-butyl((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylate(15.6 g, 34.7 mmol) in methanol (78 mL) was added 4 M HCl in dioxane (4M) (87 mL, 347 mmol) at RT. The reaction mixture was stirred for 2 h,and diisopropyl ether (156 mL) was added dropwise. The reaction mixturewas stirred for 18 h, filtered, washed with diisopropyl ether (20 mL)and dried at 50° C. under vacuum for 2 h to give the 3wHCl salt of thetitle intermediate (11.33 g, 71.2% yield). HPLC Method 3 Retention time9.87 min.

(d)3-((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)propanenitrile(crude)

To a mixture of the product of the previous step (11.24 g, 24.50 mmol),DMF (56.2 mL), and methanol (5.75 mL) was added DBU (15 mL, 103 mmol)dropwise at RT followed by acrylonitrile (2.4 mL, 36.7 mmol) dropwise.The reaction mixture was stirred at RT for 3 h and then 2:1methanol:water (225 mL) was added dropwise over 1 h. After 3 h, thereaction mixture was filtered, washed with 1:1 methanol:water (20 mL),and dried at 50° C. under vacuum overnight to provide the title compound(9.42 g, 96% yield).

(e)3-((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)propanenitrile

To a mixure of crude3-((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)propanenitrile(38.6 g, 96 mmol) was added DMF (232 mL) followed by SiliaMetS® Thiolfunctionalized silica (1.41 mmol/g, 6.8 g). The mixture was warmed to75° C. and stirred for 45 min at 75° C. The mixture was cooled to 25°C., filtered, rinsed with DMF (1 mL), and then 2:1 MeOH:water (926 mL)was added to the filtrate dropwise. The mixture was stirred at RTovernight, filtered, washed with 1:1 MeOH:water (40 mL) and dried at 50°C. under vacuum to give the title compound as a crystalline solvate (38g, 94 mmol, 98% yield) HPLC Method 3 Retention time 10.23 min. Residualsolvents by gas chromatography: methanol 6.6%, N,N-dimethylformamide2.3%, water by Karl Fischer analysis 1.2%.

Example 19 Crystalline3-((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)propanenitrileForm I (a)N⁵-((1R,3s,5S)-8-azabicyclo[3.2.1]octan-3-yl)-N⁷-(5-methyl-1H-pyrazol-3-yl)-1,6-naphthyridine-5,7-diamine

To a reactor was added the 3HCl salt ofN⁵-((1R,3s,5S)-8-azabicyclo[3.2.1]octan-3-yl)-N⁷-(5-methyl-1H-pyrazol-3-yl)-1,6-naphthyridine-5,7-diamine(3.4 kg, 1 equiv), followed by water (34 kg, 10 equiv) and 1N HCl (7 kg,2.05 equiv) to form a reaction mixture. Activated charcoal (0.22 kg,0.064 equiv) was added, followed by SiliaMetS® thiol functionalizedsilica (1.7 kg, 0.5 equiv) and the reaction mixture was agitated at 80°C. for 16 h, cooled to 25° C., and filtered through a pad of Celite toNalgene containers. The reactor was washed with water (13.6 kg, 4equiv), which was transferred to wash the cake on the filter andcollected in the Nalgene containers.

To the collected wash was added methanol (13.6 kg, 4 equiv). Thetemperature was adjusted to 20° C. and 30% w/v NaOH (4.4 kg, 1.29 equiv)was added slowly maintaining the temperature below 30° C. The resultingslurry was agitated for 3 h at 25° C., filtered, washed with water (17kg, 5 equiv), and dried at 50° C. in vacuo for 12 h to provide the titleintermediate (2.3 kg, HPLC purity 98.4%) HPLC Method 3 with 32 mingradient (time(min)/% B): 0/2, 10/20, 24/90, 27/90, 27.1/2, 32/2Retention time 9.4 min

(b)3-((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)propanenitrile(crude)

To a reactor was added the product of the previous step (2.1 kg, 1equiv), DMF (19.7 kg, 9.4 equiv), methanol (3.4 kg, 1.6 equiv), THF (9.5kg, 4.5 equiv), and DBU (0.92 kg, 0.44 equiv) and the reaction mixturewas agitated at 30° C. until fully dissolved. The temperature wasadjusted to 20° C., acrylonitrile (0.48 kg, 0.23 equiv) was added, thereaction mixture was agitated for 16 h, water (52.5 kg, 25 equiv) wasadded, and the temperature was adjusted to 20° C. The resulting slurrywas agitated for 3 h, filtered, washed with methanol (3.4 kg, 1.5 equiv)which had first washed the reactor, and dried at 50° C. in vacuo for 12h. Ethanol (21 kg, 10 equiv) was added to the dry cake, and theresulting slurry was agitated at reflux for 4 h, cooled to 25° C.,agitated at 25° C. for 1 h, and filtered. The wet cake was washed withethanol (3.2 kg, 1.5 equiv) and dried at 50° C. in vacuo for 12 h toprovide the title intermediate (1.8 kg, HPLC purity 99.8%)

(c) Crystalline solvate3-((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)propanenitrileForm II

To a reactor was added the product of the previous step (1.5 kg, 1equiv) and DMF (8.4 kg, 5.6 equiv) and the reaction mixture was heatedto 25° C. and agitated for 5 min until fully dissolved. Methanol (14.3kg, 9.5 equiv) and water (9.0 kg, 6 equiv) were added over 1 h. Theresulting slurry was agitated for 16 h at 25° C., filtered, and washedwith methanol (3.0 kg, 2 equiv) which had first washed the reactor, anddried at 50° C. in vacuo for 12 h to provide the title intermediate (1.4kg, HPLC purity 99.8%).

(d) Crystalline3-((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)propanenitrileForm I

[Equivalents measured with respect to step (c).] To a reactor was addedthe product of the previous step (1.4 kg) followed by acetone (13.8 kg,9.2 equiv) and the resulting slurry was agitated for 18 h at 45° C.,cooled to 25° C., agitated at 25° C. for 30 min, filtered, washed withacetone (3.0 kg, 2 equiv) which had first washed the reactor, and driedat 50° C. in vacuo for 12 h to provide the title compound (1.2 kg, HPLCpurity 99.8%) as a yellow crystalline solid. HPLC Column AgilentPoroshell EC C-18 150×4.6 mm, 2.7 μm, 45° C., 2.2 mL/min, 7 μL, 250 nmdetection, Mobile Phase A: Water:ACN:TFA (99:1:0.1), Mobile Phase B:Water:ACN:TFA (10:90:0.1) Gradient 37 min (time (min)/% B) 0/4, 25/27,30/100, 33/100, 33.1/4, 37/4 Retention Time 11.2 min. ¹H NMR (d₆-DMSO,600 mHz) δ (ppm) 11.75 (s, 1H), 8.76 (s, 1H), 8.57 (d, J=3 Hz, 1H), 8.41(d, J=3 Hz, 1H), 7.15 (d, J=5 Hz, 1H), 6.96 (dd, J=3 Hz, 5 Hz, 1H), 6.67(s, 1H), 6.20 (s, 1H), 4.55 (m, 1H), 3.33 (m, 2H), 2.63 (m,4H), 2.22 (s,3H), 1.70-1.93 (m, 8H).

Example 20 Crystalline3-((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)propanenitrileForm I (a) Crystalline solvate3-((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)propanenitrileForm II

To a mixture ofN⁵-((1R,3s,5S)-8-azabicyclo[3.2.1]octan-3-yl)-N⁷-(5-methyl-1H-pyrazol-3-yl)-1,6-naphthyridine-5,7-diamine(5 g, 14.31 mmol) and DMF (50 mL) was added DBU (5.39 mL, 35.8 mmol)followed by 3-bromopropionitrile (1.78 mL, 21.5 mmol) dropwise. Thereaction mixture was stirred at 20-25° C. for 4 h and then 3:1methanol:water (150 mL) was added dropwise over 60 min. The reactionmixture was stirred for 20 h at 20° C., filtered, washed with 3:1methanol:water (10 mL), and dried in vacuo at 50° C. for 2 h to providethe title compound (5.07 g, 12.60 mmol, 88% yield) HPLC Method 3Retention time 10.13 min.

To a mixture of the product of the previous step (1 g, 2.49 mmol) in DMF(6 mL) was added 3:1 methanol:water (18 mL) dropwise. After 3 h, themixture was filtered, washed with methanol (2 mL), and dried in vacuo at50° C. for 18 h to provide the title compound (0.94 g, 2.33 mmol, 94%yield) HPLC Method 3 Retention time 10.17 min.

(b) Crystalline3-((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)propanenitrileForm I

A mixture of the product of the previous step (0.6 g, 1.49 mmol) andacetone (7.2 mL) was stirred at RT for 18 h, filtered, and washed withacetone to give the title compound (0.5 g, 1.24 mmol, 83% yield). HPLCMethod 3 Retention time 10.03 min.

Example 21 Crystalline3-((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)propanenitrileForm I

A mixture of3-((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)propanenitrilesolvate Form II (100 g, 248 mmol) and 1,4-dioxane (1200 mL) was heatedto 95° C., stirred for 10 h, cooled to RT, stirred for 3 h, filtered,washed with dioxane, and dried at 50° C. for 6 h and then at RT for 5days to provide the title compound (87 g, 86% yield) HPLC Method 3Retention time 10.21 min.

Example 22 Crystalline3-((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)propanenitrileForm I

A mixture of3-((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)propanenitrilesolvate Form II (5 g, 12.42 mmol) and toluene (75 mL) was heated toreflux for 4 h, cooled to RT over 30 min, stirred at RT for 30 min,filtered, and washed with toluene to provide the title compound (4.6 g,92% yield).

Example 23 Crystalline3-((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)propanenitrileForm I

A mixture of3-((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)propanenitrileethanol solvate (1 g, 2.49 mmol) and butyl acetate (20 mL) was heated to110° C. for 8 h, cooled to RT, stirred at RT for 65 h, filtered, andwashed with water to provide the title compound (0.92 g, 92% yield).HPLC Method 3 Retention time 10.08 min.

Examples 24-27 Properties of the Solid Forms of the Invention

Samples of the Form I crystalline freebase and Form II crystallinesolvate of3-((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)propanenitrileof Examples 19 and 18, respectively, were analyzed by powder X-raydiffraction (PXRD). Crystalline Form I of Example 19 was also analyzedby differential scanning calorimetry (DSC), thermogravimetric analysis(TGA), dynamic moisture sorption (DMS), and by single crystal x-raydiffraction.

Example 24 Powder X-Ray Diffraction

The powder X-ray diffraction patterns of FIGS. 1 and 5 were obtainedwith a Bruker D8-Advance X-ray diffractometer using Cu-Kα radiation(λ=1.54051 Å) with output voltage of 45 kV and current of 40 mA. Theinstrument was operated in Bragg-Brentano geometry with incident,divergence, and scattering slits set to maximize the intensity at thesample. For measurement, a small amount of powder (5-25 mg) was gentlypressed onto a sample holder to form a smooth surface and subjected toX-ray exposure. The samples were scanned in 20-20 mode from 2° to 40° in20 with a step size of 0.02° and a scan speed of 0.30° seconds per step.The data acquisition was controlled by Bruker DiffracSuite measurementsoftware and analyzed by Jade software (version 7.5.1). The instrumentwas calibrated with a corundum standard, within ±0.02° two-theta angle.Observed PXRD two-theta peak positions and d-spacings are shown inTables 9 and 10, respetively for crystalline Form I and the crystallineForm II solvate.

TABLE 9 PXRD Data for Crystalline Form I 2-Theta d(Å) Area A % 7.8711.23 515.2 26.3 10.80 8.18 188.7 9.6 12.78 6.92 731.1 37.3 12.86 6.88620.9 31.7 13.47 6.57 423.0 21.6 13.64 6.49 274.5 14.0 14.66 6.04 105.25.4 15.11 5.86 285.6 14.6 15.54 5.70 541.5 27.6 15.78 5.61 1959.5 100.017.75 4.99 356.7 18.2 18.00 4.92 201.7 10.3 20.41 4.35 944.3 48.2 20.544.32 938.4 47.9 21.00 4.23 303.1 15.5 22.22 4.00 502.1 25.6 22.79 3.90158.5 8.1 22.93 3.88 272.8 13.9 23.49 3.79 639.2 32.6 23.65 3.76 1685.486.0 23.75 3.74 903.8 46.1 25.23 3.53 115.0 5.9 25.31 3.52 101.7 5.226.36 3.38 78.9 4.0 27.26 3.27 115.7 5.9 27.44 3.25 134.1 6.8

TABLE 10 PXRD Data for the Crystalline Form II solvate 2-Theta d(Å) AreaA % 6.97 12.67 941.9 16.4 9.76 9.05 5759.9 100.0 10.17 8.69 803.6 14.011.56 7.65 531.9 9.2 12.68 6.97 1058.9 18.4 13.62 6.50 441.4 7.7 13.986.33 472.6 8.2 15.06 5.88 1170.0 20.3 16.61 5.33 2410.1 41.8 17.27 5.13670.0 11.6 19.02 4.66 786.5 13.7 20.40 4.35 1472.6 25.6 21.04 4.221062.2 18.4 21.20 4.19 1578.9 27.4 21.99 4.04 5002.2 86.8 22.44 3.961663.1 28.9

Example 25 Thermal Analysis

Differential scanning calorimetry (DSC) was performed using a TAInstruments Model Q-100 module with a Thermal Analyst controller. Datawere collected and analyzed using TA Instruments Thermal Analysissoftware. A sample of each crystalline form was accurately weighed intoa covered aluminum pan. After a 5 minute isothermal equilibration periodat 5° C., the sample was heated using a linear heating ramp of 10°C./min from 0° C. to 250° C. A representative DSC thermogram of the FormI crystalline freebase of the invention is shown in FIG. 2.

Thermogravimetric analysis (TGA) measurements were performed using a TAInstruments Model Q-50 module equipped with high resolution capability.Data were collected using TA Instruments Thermal Analyst controller andanalyzed using TA Instruments Universal Analysis software. A weighedsample was placed onto a platinum pan and scanned with a heating rate of10° C. from ambient temperature to 300° C. The balance and furnacechambers were purged with nitrogen flow during use. A representative TGAtrace of the Form I crystalline freebase of the invention is shown inFIG. 3.

Example 26 Dynamic Moisture Sorption Assessment

Dynamic moisture sorption (DMS) measurement was performed using a VTIatmospheric microbalance, SGA-100 system (VTI Corp., Hialeah, Fla.33016). A weighed sample was used and the humidity was lowest possiblevalue (close to 0% RH) at the start of the analysis. The DMS analysisconsisted of an initial drying step (0% RH) for 120 minutes, followed bytwo cycles of sorption and desorption with a scan rate of 5% RH/stepover the humidity range of 5% RH to 90% RH. The DMS run was performedisothermally at 25° C. A representative DMS trace for the Form Icrystalline freebase of the invention is shown in FIG. 4.

Example 27 Single Crystal X-Ray Diffraction

Intensity data were collected at 293° K, using Cu radiation (1=1.54184Å), on an Oxford Diffraction Gemini-R Ultra diffractometer operated bythe CrysAlis software. [Agilent Technologies (2012), Yarnton, England](CrysAlis CCD and CrysAlis RED, 2003) The data were corrected forabsorption effects by means of comparison of equivalent reflections. Thestructure was solved with the direct methods procedure implemented inSHELXT and refined by full-matrix least squares on F² using SHELXL-2014.[Sheldrick, Acta Cryst. C71 (2015), 3-8]. Non-hydrogen atoms werelocated in difference maps and refined anisotropically. Hydrogen atomsbonded to C atoms were fixed in idealized positions. Their thermaldisplacement parameters were refined freely, except for one of themethyl groups. H atoms bonded to N were refined using distancerestraints, N—H=0.86(1) Å, and their thermal displacement parameterswere refined freely.

Example 28 Solid State Stability Assessment

Samples of the Form I crystalline freebase of the invention were storedin zip-tie closed double polyethylene bags inside an HDPE bottle withscrew cap at 25° C. and 60% relative humidity (RH) and at 40° C. and 75%RH. At specific intervals, the contents of a representative sample wasremoved and analyzed by by HPLC for chemical purity and by Karl Fischerfor water content.

TABLE 11 Crystalline Form I Stability Study T = 1 Month T = 3 Month T =3 Month 40° C./ 40° C./ 25° C./ T = 0 75% RH 75% RH 60% RH HPLC puritya/a % 99.76 99.79 99.75 99.77 Assay % w/w 97.4 98.7 93.8 96.3 Water (KF)% w/w <0.2 NT^(a) <0.2 NT^(a) RRT 0.96 0.07 0.07 0.07 0.07 1.84 0.080.08 0.05 0.05 ^(a)NT = not testedBiological Assays

The compounds of the invention have been characterized in one or more ofthe following biological assays.

Assay 1: Biochemical JAK and Off-target Kinase Assays

A panel of four LanthaScreen JAK biochemical assays (JAK1, 2, 3 andTyk2) were carried in a common kinase reaction buffer (50 mM HEPES, pH7.5, 0.01% Brij-35, 10 mM MgCl2, and 1 mM EGTA). Recombinant GST-taggedJAK enzymes and a GFP-tagged STAT1 peptide substrate were obtained fromLife Technologies.

Serially diluted compounds were pre-incubated with each of the four JAKenzymes and the substrate in white 384-well microplates (Corning) atambient temperature for lh. ATP was subsequently added to initiate thekinase reactions in 10 μL total volume, with 1% DMSO. The final enzymeconcentrations for JAK1, 2, 3 and Tyk2 are 4.2 nM, 0.1 nM, 1 nM, and0.25 nM respectively; the corresponding Km ATP concentrations used are25 μM, 3 μM, 1.6 μM, and 10 μM; while the substrate concentration is 200nM for all four assays. Kinase reactions were allowed to proceed for 1hour at ambient temperature before a 10 μL preparation of EDTA (10 mMfinal concentration) and Tb-anti-pSTAT1 (pTyr701) antibody (LifeTechnologies, 2 nM final concentration) in TR-FRET dilution buffer (LifeTechnologies) was added. The plates were allowed to incubate at ambienttemperature for lh before being read on the EnVision reader (PerkinElmer). Emission ratio signals (520 nm/495 nm) were recorded andutilized to calculate the percent inhibition values based on DMSO andbackground controls.

For dose-response analysis, percent inhibition data were plotted vs.compound concentrations, and IC50 values were determined from a4-parameter robust fit model with the Prism software (GraphPadSoftware). Results were expressed as pIC50 (negative logarithm of IC50)and subsequently converted to pKi (negative logarithm of dissociationconstant, Ki) using the Cheng-Prusoff equation.

Test compounds having a higher pKi value in each of the four JAK assaysshow greater inhibition of JAK activity. Compounds of the inventiontested in this assay typically exhibited pKi values between about 7 andabout 10.3

A panel of off-target tyrosine kinase assays (Flt3, RET, FGFR2, TrkA,and pDGFRβ) were developed using a similar methodology, with recombinantenzymes obtained from Life Technologies and biotinylated peptidesubstrates synthesized at AnaSpec. All assays were carried out atambient temperature with a final ATP concentration of 100 μM. Detectionreagents, including Eu-anti-phosphotyrosine (pY20) antibody andSureLight APC-SA, were purchased from Perkin Elmer. Emission ratiosignals (665 nm/615 nm) were recorded and utilized for data analysis,and the final results were expressed as pIC₅₀.

Assay 2: Cellular JAK Potency Assay

The AlphaScreen JAM cellular potency assay was carried out by measuringinterleukin-13 (IL-13, R&D Systems) induced STAT6 phosphorylation inBEAS-2B human lung epithelial cells (ATCC). The anti-STAT6 antibody(Cell Signaling Technologies) was conjugated to AlphaScreen acceptorbeads (Perkin Elmer), while the anti-pSTAT6 (pTyr641) antibody (CellSignaling Technologies) was biotinylated using EZ-Link Sulfo-NHS-Biotin(Thermo Scientific).

BEAS-2B cells were grown at 37° C. in a 5% CO₂ humidified incubator in50% DMEM/50% F-12 medium (Life Technologies) supplemented with 10% FBS(Hyclone), 100 U/mL penicillin, 100 μg/mL streptomycin (LifeTechnologies), and 2 mM GlutaMAX (Life Technologies). On day 1 of theassay, cells were seeded at a 7,500 cells/well density in whitepoly-D-lysine-coated 384-well plates (Corning) with 254 medium, and wereallowed to adhere overnight in the incubator. On day 2 of the assay, themedium was removed and replaced with 12 μL of assay buffer (Hank'sBalanced Salt Solution/HBSS, 25 mM HEPES, and 1 mg/ml bovine serumalbumin/BSA) containing dose-responses of test compounds. Compounds wereserially diluted in DMSO and then diluted another 1000-fold in media tobring the final DMSO concentration to 0.1%. Cells were incubated withtest compounds at 37° C. for 1 h, and followed by the addition of 12 μLof pre-warmed IL-13 (80 ng/ml in assay buffer) for stimulation. Afterincubating at 37° C. for 30 min, the assay buffer (containing compoundand IL-13) was removed, and 10 μL of cell lysis buffer (25 mM HEPES,0.1% SDS, 1% NP-40, 5 mM MgCl2, 1.3 mM EDTA, 1 mM EGTA, and supplementwith Complete Ultra mini protease inhibitors and PhosSTOP from RocheDiagnostics). The plates were shaken at ambient temperature for 30 minbefore the addition of detection reagents. A mixture ofbiotin-anti-pSTAT6 and anti-STAT6 conjugated acceptor beads was addedfirst and incubated at ambient temperature for 2 h, followed by theaddition of streptavidin conjugated donor beads (Perkin Elmer). After aminimum of 2 h incubation, the assay plates were read on the EnVisionplate reader. AlphaScreen luminescence signals were recorded andutilized to calculate the percent inhibition values based on DMSO andbackground controls.

For dose-response analysis, percent inhibition data were plotted vs.compound concentrations, and IC50 values were determined from a4-parameter robust fit model with the Prism software. Results wereexpressed as the negative logarithm of the IC₅₀ value, pIC₅₀.

Test compounds having a higher pIC₅₀ value in this assay show greaterinhibition of IL-13 induced STAT6 phosphorylation. Compounds of theinvention tested in this assay typically exhibited pIC₅₀ values betweenabout 6.8 and about 8.5.

An interleukin-4 (IL-4, R&D Systems) induced STAT6 phosphorylation assaywas also developed in THP-1 human monocytic cells (ATCC), using theidentical assay format and detection reagents. IL-4 stimulation wascarried out for 30 min at a 30 ng/ml final concentration. Datacollection and analysis were also conducted in a similar fashion.Compounds of the invention tested in this assay typically exhibitedpIC₅₀ values between about 6.8 and about 8.5.

Assay 3: Cytotoxicity Assay

A CellTiter-Glo luminescent cell viability/cytotoxicity assay wascarried out in BEAS-2B human lung epithelial cells (ATCC) under thenormal growth condition.

Cells were grown at 37° C. in a 5% CO₂ humidified incubator in 50%DMEM/50% F-12 medium (Life Technologies) supplemented with 10% FBS(Hyclone), 100 U/mL penicillin, 100 μg/mL streptomycin (LifeTechnologies), and 2 mM GlutaMAX (Life Technologies). On day 1 of theassay, cells were seeded at a 500 cells/well density in white 384-welltissue culture plates (Corning) with 25 μL medium, and were allowed toadhere overnight in the incubator. On day 2 of the assay, 5 μL of mediumcontaining dose-responses of test compounds was added, and incubated at37° C. for 48 h. 30 μl of CellTiter-Glo detection solution (Promega) wassubsequently added, mixed on an orbital shaker for 5 min, and incubatedfor additional 10 min before being read on the EnVision reader.Luminescence signals were recorded and percent DMSO control values werecalculated.

For dose-response analysis, percent DMSO control data were plotted vs.compound concentrations to derive dose-response curves by lineconnecting each data point. The concentration at which each curvecrosses the 15% inhibition threshold is defined as CC₁₅. Results wereexpressed as the negative logarithm of the CC₁₅ value, pCC₁₅.

It is expected that test compounds exhibiting a lower pCC₁₅ value inthis assay have less likelihood to cause cytotoxicity. Compounds of theinvention tested in this assay typically exhibited pCC₁₅ values betweenless than 5 and about 6.

In Vitro Assay Results

All of the compounds of Examples 1 to 17 and Tables 1 to 8 were testedin one or more of the assays described above. In the following tables,for the JAK1, JAK 2, JAK3, and TYK2 enzyme assays, A represents a pK_(i)value≥10 (K_(i)≤0.1 nM), B represents a pK_(i) value between 9 and 10(K_(i) between 1 nM and 0.1 nM), C represents a pK_(i) value between 7and 9 (K_(i) between 100 nM and 1 nM), and D represents a pK_(i) valuebetween 6.5 and 7 (K_(i) between 316 nM and 100 nM). For the THP-1 andBEAS-2B cell potency assays, A represents a pEC₅₀ value≥7.5 (EC₅₀≤32nM), B represents a pEC₅₀ value between 6.7 and 7.5 (EC₅₀ between 200 nMand 32 nM), and C represents a pEC₅₀ value<6.7 (EC₅₀>200 nM).

Example JAK 1 JAK 2 JAK 3 Tyk 2 THP-1 BEAS2B Number (pKi) (pKi) (pKi)(pKi) IL4 pIC50 pIC50 1 A A C B B B 2 B B C C B B 3 B B C C B A 4 B B CC B 5 B C C C A A 6 B B C C B B 7 A B C B A A 8 A A B B A A 9 B B C B AA 10 A A B B A A 11 A A B A B A 12 B B C C B B 13 A A C B B B 14 A A C BA A 15 B A B B A A 16 B C C C B 17 B A B B B B

TABLE 1 Example JAK 1 JAK 2 JAK 3 Tyk 2 THP-1 BEAS2B Number (pKi) (pKi)(pKi) (pKi) IL4 pIC50 pIC50 1-1 B B C C C 1-2 C C C C C 1-3 C C C C C1-4 B B C C B 1-5 B B C C B 1-6 B B C C C 1-7 B B C C C 1-8 B C C C C1-9 B C C C C 1-10 B B C C C 1-11 B B C C C 1-12 B B B C C 1-13 B C C CA 1-14 B B C B A A 1-15 C C D C C 1-16 B B C C B 1-17 B B C C B B 1-18 BB C C C 1-19 A B B C B 1-20 B B C C B 1-21 B B C C A B 1-22 B B C B B A1-23 A A B B A A 1-24 B A C B B A 1-25 B A C B A A 1-26 B B C C B 1-27 BB C C B 1-28 B C B C C 1-29 C C C C C 1-30 B A B B B 1-31 A A B B A A1-32 B A B B C 1-33 B B C C C 1-34 B C C C C 1-35 B B B B A 1-36 B B C CB 1-37 B A B C B 1-38 B B C C B 1-39 B B C C A A 1-40 B B C C A A 1-41 BA B B C 1-42 B A B C C 1-43 B B C C A A 1-44 B B C C A A 1-45 B B C B B1-46 B C C C B 1-47 B A B B A A 1-48 A A B B B 1-49 A A B B A 1-50 A A CC B 1-51 B A B B B 1-52 B B C B B 1-53 B B C C B 1-54 B A B B B 1-55 B BB C B 1-56 B B C B A A 1-57 B B C C B 1-58 A A B B B

TABLE 2 Example JAK 1 JAK 2 JAK 3 Tyk 2 THP-1 BEAS2B Number (pKi) (pKi)(pKi) (pKi) IL4 pIC50 pIC50 2-1 B C C C B B 2-2 B C C C B B 2-3 B B C CB A 2-4 C C C C C 2-5 B C C C C 2-6 B B C C B A 2-7 B B C C B 2-8 B B CC B 2-9 C C C C C 2-10 B B C C B 2-11 B B C C B 2-12 B B C C B 2-13 B BC C B 2-14 B B C C C 2-15 B B C C A 2-16 B B C B B B 2-17 B B C C B A2-18 B B C B A 2-19 B B C B A 2-20 B B C C A A 2-21 B B C C B 2-22 B B CB B A 2-23 B B C C B 2-24 B B C C A A 2-25 B A B B A A 2-26 B B C C C2-27 B B C B B 2-28 B A C B C 2-29 B B C B B 2-30 B B C C C 2-31 B B C CB

TABLE 3 Example JAK 1 JAK 2 JAK 3 Tyk 2 THP-1 BEAS2B Number (pKi) (pKi)(pKi) (pKi) IL4 pIC50 pIC50 3-1 B B C C A 3-2 B B C C A 3-3 B C C C A3-4 B B C C A 3-5 B C C C B 3-6 B A C B A 3-7 B B C C B 3-8 B B C C A3-9 B B C C A 3-10 B B C B B 3-11 B B C C A 3-12 B B C C B 3-13 B B C CB B 3-14 B B C C B A

TABLE 4 Example JAK 1 JAK 2 JAK 3 Tyk 2 THP-1 BEAS2B Number (pKi) (pKi)(pKi) (pKi) IL4 pIC50 pIC50 4-1 A B C B A 4-2 B B C C A A 4-3 B B C B AA 4-4 B B C C A A 4-5 B B C C A 4-6 B B C C A 4-7 B B C B A 4-8 B B C BA 4-9 B B C C C 4-10 B B C C B B 4-11 B C C C C 4-12 B C C C B 4-13 B CC C B A 4-14 C C C C B 4-15 B B C C A A 4-16 A A B B A 4-17 B B C B A4-18 A A B B A A 4-19 A A C B A A 4-20 B A B B A A 4-21 B B C B C 4-22 BA B B A 4-23 B A B B A A 4-24 B B C C B 4-25 B B C C A 4-26 B B C C B B4-27 B A B B A A 4-28 B B C B A 4-29 A A B B A A

TABLE 5 Example JAK 1 JAK 2 JAK 3 Tyk 2 THP-1 BEAS2B Number (pKi) (pKi)(pKi) (pKi) IL4 pIC50 pIC50 5-1 B B C B A A 5-2 B C C C B A 5-3 B C C CB 5-4 B C C C A A 5-5 B C C C B 5-6 B B C C A A 5-7 B C C C B A 5-8 B BC B A A 5-9 B B C B A A 5-10 A A B B A A 5-11 B A B B A B 5-12 A A C B B5-13 A A C B B 5-14 B B C C B B 5-15 B A B B B 5-16 B A B B B B 5-17 B BB B B A 5-18 B A B B B 5-19 B C C C B 5-20 B B C B A B 5-21 B A B B A A5-22 A A B B B A 5-23 A A B B A 5-24 B B B B B 5-25 B B B B B 5-26 A A AB A 5-27 B B B B A A 5-28 B B B B B 5-29 B A B B B 5-30 B A B B A A 5-31B B C C B 5-32 B B B B B 5-33 B B B B B 5-34 B B C B B 5-35 B B B B A5-36 B A B B A A 5-37 B B B B B 5-38 C C C C 5-39 B B C B B 5-40 B B C CB 5-41 B B C B C 5-42 B B C C C 5-43 B B C B B

TABLE 6 Example JAK 1 JAK 2 JAK 3 Tyk 2 THP-1 BEAS2B Number (pKi) (pKi)(pKi) (pKi) IL4 pIC50 pIC50 6-1 B B C B A A 6-2 B B C C A B 6-3 B B C CB B 6-4 B A C B A A 6-5 B A C B A A 6-6 B A B B A A 6-7 C B C C B 6-8 BA B B A A 6-9 B A B B A A 6-10 B B C B B 6-11 B B C B A 6-12 B B C C B6-13 B C C C B 6-14 B C C C A A 6-15 B B C C A A 6-16 B C C C B B 6-17 BC C C A A 6-18 A A C B A A 6-19 B C C C B 6-20 B B C B B 6-21 B B C B B6-22 B B C B B 6-23 B A C B B 6-24 B B C C B B 6-25 C C C C B A 6-26 B CC C B 6-27 B B C C B 6-28 B B C C A A 6-29 B B C B A A 6-30 B B C B C6-31 B B C B A A 6-32 A A C B A 6-33 A A C B B 6-34 A A C B A 6-35 B A CB B 6-36 B B C C B 6-37 B B C C B 6-38 B B C C B 6-39 B C C C C 6-40 B BC C A 6-41 A B C C A B 6-42 B B C C C 6-43 B B C C B 6-44 B C C C B 6-45B B C C B 6-46 B B C C C 6-47 A A B B A A 6-48 B B C C B 6-49 B B C C B6-50 A A B B C 6-51 B B C C C 6-52 B B C C C 6-53 B B C C C 6-54 B B C CC 6-55 C C C C C 6-56 B A C B B B 6-57 B A C C C 6-58 B A B C C 6-59 B AB B C 6-60 A A B B B

TABLE 7 Example JAK 1 JAK 2 JAK 3 Tyk 2 THP-1 BEAS2B Number (pKi) (pKi)(pKi) (pKi) IL4 pIC50 pIC50 7-1 B C C C B 7-2 B B C C A A 7-3 C C C C C7-4 B C C C B B 7-5 B C C C B B 7-6 B B C C A B 7-7 C C C C B 7-8 B B CC B 7-9 B B C C A A 7-10 B B C B A B

TABLE 8 Example JAK 1 JAK 2 JAK 3 Tyk 2 THP-1 BEAS2B Number (pKi) (pKi)(pKi) (pKi) IL4 pIC50 pIC50 8-1 B B C C B 8-2 B B C C A C 8-3 B B C C A8-4 B C C C B B 8-5 B C C C B A 8-6 B B C C B B 8-7 C C C C C 8-8 C C CC C 8-9 B B C C B 8-10 A A C B A A 8-11 B B C C B A 8-12 B B C B A A8-13 B B C C B A 8-14 B B C C A A 8-15 B C C C C 8-16 B B C C B 8-17 A AC C B 8-18 B B C C B 8-19 B B C C C 8-20 B C D C C 8-21 B B C C B 8-22 BB C C B B 8-23 B A C B B B 8-24 B B C C B B

Assay 4: Determination of Absorption in Cannulated Rats

Oral bioavailability (F %), fraction absorbed (F_(a)%) and fractionescaping hepatic clearance (F_(h)%) were determined in Sprague Dawleyrats from the following two studies:

(1) Pharmacokinetics in rats following an IV dose of test compound:Following IV dosing, plasma samples were typically collected from 0-6hr. Drug levels were determined using an LC-MS-MS method. The resultingdrug levels were used to compute the IV pharmacokinetic parameters: AUCIV and Dose IV.

(2) Rats that have been cannulated in their portal vein (PV) and also intheir jugular vein (JV) were dosed orally with test compound. Followingoral dosing, plasma samples were typically collected from 0-6 hr fromboth the portal vein and the jugular vein. Drug levels were determinedusing an LC-MS-MS method. The resulting drug levels were used to computethe following pharmacokinetic parameters: AUC PO PV, AUC PO JV, and DosePO.

Using data derived from the above studies, the oral bioavailability F %,and the quantities F_(a)% and F_(h)% were calculated from the followingformulas:F %=(AUC PO JV/AUC IV)*(Dose IV/Dose PO)*100F _(a)%=(AUC PO PV/AUC IV)*(Dose IV/Dose PO)*100F _(h)%=AUC PO JV/AUC PO PV

where:

AUC PO JV=Area under the curve following oral dose and plasma collectedfrom the jugular vein

AUC PO PV=Area under the curve following oral dose and plasma collectedfrom the portal vein

AUC IV=Area under the curve following an intravenous dose

Dose IV=Intravenous Dose in mg/kg

Dose PO=Oral Dose in mg/kg

Compounds of the invention typically exhibited oral bioavailability (F%) less than about 10% and absorption at the portal vein (F_(a)%) lessthan about 20% including less than about 10%. For example the compoundsof Examples 1-6, 8, and 12 all exhibited F % values less than about 5%and F_(a)% values less than about 10%.

Assay 5: Colon Pharmacokinetics in Rats

Test compounds were individually formulated in 0.5% methyl-cellulose inwater and dosed via oral gavage at 5 mg/kg to Sprague Dawley rats. Atvarious time points (typically 1, 2, 4, 6, 24 hr) post dosing, bloodsamples were removed via cardiac puncture and intact colons were excisedfrom the rats. Blood samples were centrifuged at 1500×g for 15 min tocollect plasma. Colons were washed with ice cold phosphate bufferedsaline (PBS), weighed, and homogenized at a dilution of 1:10 in PBS.Plasma and colon levels of test compound were determined by LC-MSanalysis against analytical standards constructed into a standard curvein the test matrix. A colon to plasma ratio was determined as the ratioof the colon AUC to the plasma AUC in μg hr/g. For example, thecompounds of examples 1, 2, and 5 exhibited a colon to plasma ratio inexcess of about 450.

Assay 6: Pharmacokinetics in Plasma and the GI Tract in Rats and Dogs

A test compound was dosed to male Sprague Dawley rats (n=3) as describedin Assay 5. At each time point (0.5, 1, 3, 6, and 24 hr) plasma sampleswere taken by cardiac puncture and immediately afterward the GI tractwas removed and the following segments excised: duodenum, proximalcolon, and distal colon. Plasma and segment levels of test compound weredetermined as described in Assay 5. At all time points, the plasmaconcentration was below the limit of quantitation of 0.001 μg/mL. Forthe compound of example 1, for each segment, the tissue to plasma ratiowas in excess of about 14000.

An analogous experiment was performed in dogs. Male beagle dogs (n=2)were dosed via oral gavage with 5 mg/kg of test compound, formulated asabove. In dog number 1, plasma samples were taken at 0.25, 1, 2, 4, 6,and 24 hr post dosing. In dog number 2, plasma samples were taken to 6hr. In both dog number 1 (at 24 hr) and dog number 2 (at 6 hr) the GItract was removed and segmented as follows: duodenum, ileum, cecum andthe colon segmented into equal thirds (proximal, middle, and distalcolon). For each of the GI segments, an approximately 2 cm piece wasexcised in the middle of the segment. Each was washed thoroughly in icecold PBS buffer and then homogenized in 5 volumes of PBS buffer andanalyzed as above. For the compound of example 1, the ratio ofconcentration of compound in GI tissue to compound in plasma forcollections conducted at 6 hours post oral dose ranged from about 9 toabout 165 where the colon concentration was taken as the sum of thethree colon segments. The GI tissue to plasma ratio ranged from about 7to about 30 for collections conducted at 24 hours post oral dose.

Assay 7: Mouse Model of Oxazalone-Induced Colitis

Oxazolone-induced colitis is an experimental model that has ahistological resemblance to human ulcerative colitis (Heller et al.Immunology, 2002, 17, 629-638). Adult BALB/C mice from Harlan were usedin the assay. On day 1, animals were lightly anesthetized withisoflurane and the hairs between the shoulder were carefully removedbefore oxazolone (4%, 150 μL, 4:1 acetone:olive oil formulation) orvehicle solution was slowly applied for skin sensitization. Seven daysafter skin sensitization, the mice were fasted overnight, anesthetizedwith isoflurane inhalation, and a 1 mL syringe equipped with a 3.5-Fcatheter, filled with oxazolone solution, was inserted carefully about 4cm into the colon of the mouse. Following insertion, 50 μL of theoxazolone solution (1%, 1:1 ethanol:water formulation) was injected veryslowly (over 30 sec using an injection pump) into the colon. Thecatheter was removed and the mice were held vertically (head down) for 2min to ensure that the entire oxazolone solution remained inside thecolon. Drug treatment (PO, BID or TID) or vehicle was initiated a dayprior to the oxazolone intrarectal (IR) challenge. Two-day postoxazolone intrarectal challenge, the Disease Activity Index (DAI) wasassessed by treatment-blinded experimenters for each mouse according tothe criteria score: stool consistency score (0, normal; 2, loose; 4,diarrhea), gross bleeding score (0, absence; 2, blood tinged; 4,presence), and weight loss score (0, none; 1, 1%-5%; 2, 5%-10%; 3,10%-20%; 4, more than 20%); DAI=average of (stool consistencyscore+gross bleeding score+weight loss score).

Selected compounds of the invention were tested in the assay. Efficacyin the model is evidenced by a decrease in DAI score as compared withthe score from vehicle treated animals. The compounds of examples 1, 2,3, 4, 5, 6, 8, 12, and 1-38, exhibited a statistically significantdecrease in DAI score as compared with vehicle treated animals in theoxazalone model at a dose of 1, 3, and/or 10 mg/kg BID, while thecompounds of examples 7, 9, 11, 13, 2-1, 2-6, 2-16, 2-17, 2-22, 2-24,4-3, 4-4, 4-13, 4-18, 4-19, 4-23, and 5-11 did not exhibit astatistically significant decrease at the doses up to 10 mg/kg BIDtested in the assay.

Assay 8: Immunosuppression Effects in Mouse Splenic Natural Killer (NK)Cells

Depletion of mouse splenic cells is an experimental model ofimmunosuppression (Kudlacz et al., Am. J. of Transplantation, 2004, 4,51-57). The compound of Example 1 was assessed in the mouse splenic cellmodel following the same treatment paradigm as that used in theoxazolone-induced colitis model (Assay 7).

Adult male Balb/C mice (12-14 weeks of age) from Harlan were used forthe study. The compound (1, 10 and 100 mg/kg, BID) and tofacitinib (30mg/kg, BID) as a positive control were dosed orally for three days tonaïve mice. Spleens were harvested 1 or 2 h post last dose and crushedimmediately for cell subtype staining. Prior to fixation,fluorophore-labelled antibodies for CD19 (FITC; B cells), CD3e (PE; panT cells) and DX5 (APC; NK cells) were incubated with splenocyte samplesfrom each animal to allow for simultaneous, multiple subtype % analysison the flow cytometer. The number of total spleen cells for each animalwas measured by Scepter™ 2.0 Handheld Automated Cell Counter.

The absolute number of lymphocyte subtype population (e.g., splenic B, Tand NK cells) was calculated from the percentage of each subtype timestotal spleen cells for each animal. A one way ANOVA, with Dunnett's posthoc test, was used to compare the splenic lymphocytes number of thevehicle and test compound groups. The a level was set at p<0.05. Datawere presented as the mean±SEM for each group.

The positive control, tofacitinib (30 mg/kg; PO, BID), dose-dependentlyand significantly decreased splenic NK cell counts. In the same study,splenic NK cell counts were unaffected by the compound of Example 1 atPO (BID) doses up to 100 mg/kg (the maximum dose tested). No treatmenteffect was observed for the B and T cell populations with eithercompound.

This data, in conjunction with the 1 mg/kg minimal dose that caused asignificant anti-colitic effect in the mouse model of oxazolone-inducedcolitis (Assay 7), allow a functional therapeutic index of >100 to becomputed for the compound of Example 1.

Assay 9: First in Human Study to Evaluate the Safety, Tolerability andPharmacokinetics in Healthy Subjects

The compound of Example 1 was evaluated in a double-blinded, randomized,placebo-controlled, single ascending dose (SAD) and multiple ascendingdose (MAD) study of safety, tolerability, and pharmacokinetics inhealthy subjects. Pharmacokinetic samples were collected up to 72 hourspost the final dose in both the SAD study (after the first dose) and MADstudy (after 14 days of once-daily dosing). The SAD study enrolled 5cohorts and the MAD study enrolled 4 cohorts totaling 72 subjects, ofwhich 71 completed the dosing period.

Plasma pharmacokinetic (PK) parameters were determined bynon-compartmental analysis using WinNonLin Version 6.4.0 (Pharsight, StLouis, Mo.). The plasma PK parameter presented here is:

C_(max): maximum concentration in plasma

Following a single dose up to 1000 mg, average C_(max) plasmaconcentrations of the compound were less than 50 ng/mL with noindividual subject achieving a C_(max) of greater than 100 ng/mL.Following 14 days of compound administration up to 300 mg, averageC_(max) plasma compound concentrations were less than 15 ng/mL with noindividual subject achieving a C_(max) of greater than 30 ng/mL.Comparison of these data to other orally administered compounds,suggests that the compound of Example 1 has a very low oralbioavailability. Also, high drug concentration was observed in stoolsamples suggesting significant exposure in the gastrointestinal tract.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto. Additionally, all publications, patents, andpatent documents cited hereinabove are incorporated by reference hereinin full, as though individually incorporated by reference.

What is claimed is:
 1. A crystalline form of3-((1R,3s,5S)-3-((7-((5-methyl-1H-pyrazol-3-yl)amino)-1,6-naphthyridin-5-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)propanenitrile,wherein the crystalline form is characterized by a powder X-raydiffraction pattern comprising diffraction peaks at 2θ(°) values of7.87±0.20, 12.78±0.20, 15.78±0.20, and 20.41±0.20.
 2. The crystallineform of claim 1, wherein the crystalline form is further characterizedby a powder X-ray diffraction pattern comprising two or more additionaldiffraction peaks at 2θ(°) values selected from the group consisting of10.80±0.20, 13.47±0.20, 13.64±0.20, 14.66±0.20, 15.11±0.20, 15.54±0.20,17.75±0.20, 21.00±0.20, 22.22±0.20, 22.93±0.20, and 23.65±0.20.
 3. Thecrystalline form of claim 1, wherein the crystalline form ischaracterized by a powder X-ray diffraction pattern in which the peakpositions are in accordance with the peak positions of the pattern shownin FIG.
 1. 4. The crystalline form of claim 1, wherein the crystallineform is characterized by a differential scanning calorimetry tracerecorded at a heating rate of 10° C. per minute which shows a maximum inendothermic heat flow at a temperature between 243° C. and 253° C. 5.The crystalline form of claim 4, wherein the crystalline form ischaracterized by a differential scanning calorimetry trace in accordancewith that shown in FIG.
 2. 6. A pharmaceutical composition comprisingthe crystalline claim 1 and a pharmaceutically-acceptable carrier. 7.The pharmaceutical composition of claim 6, wherein the pharmaceuticalcomposition further comprises one or more additional therapeutic agents.8. A method of inhibiting Janus kinase activity in a mammal, the methodcomprising administering to the mammal the crystalline form of claim 1.9. The method of claim 8, wherein the mammal suffers from agastrointestinal inflammatory disease.
 10. The method of claim 9,wherein the gastrointestinal inflammatory disease is selected from thegroup consisting of collagenous colitis, lymphocytic colitis, Behcet'sdisease, ileitis, eosinophilic esophagitis, and infectious colitis. 11.The method of claim 9, wherein the gastrointestinal inflammatory diseaseis ulcerative colitis.
 12. The method of claim 11, wherein theulcerative colitis is selected from the group consisting ofproctosigmoiditis, pancolitis, ulcerative proctitis and left-sidedcolitis.
 13. The method of claim 9, wherein the gastrointestinalinflammatory disease is checkpoint cancer treatment-induced colitis. 14.The method of claim 9, wherein the gastrointestinal inflammatory diseaseis cytotoxic T-lymphocyte-associated protein 4 inhibitor-inducedcolitis.
 15. The method of claim 9, wherein the gastrointestinalinflammatory disease is graft versus host disease-related colitis. 16.The method of claim 9, wherein the gastrointestinal inflammatory diseaseis Crohn's disease.
 17. The method of claim 9, wherein thegastrointestinal inflammatory disease is gastrointestinal adverseeffects in graft versus host disease.
 18. The method of claim 9, whereinthe gastrointestinal inflammatory disease is celiac disease.
 19. Themethod of claim 8, wherein the method further comprises administeringone or more additional therapeutic agents.